Human monoclonal antibodies to epidermal growth factor receptor

ABSTRACT

In accordance with the present invention, there are provided fully human monoclonal antibodies against human epidermal growth factor receptor (EGF-r). Nucelotide sequences encoding and amino acid sequences comprising heavy and light chain immunoglobulin molecules, particularly sequences corresponding to contiguous heavy and light chain sequences from CDR1 through CDR3, are provided. Hybridomas expressing such immunoglobulin molecules and monoclonal antibodies are also provided. Also provided in accordance with the invention are antibodies that possess one or more of the following functional characteristics: (i) inhibit tyrosine phosphorylation of EGF-r, (ii) do not inhibit EGF-r internalization, (ii) inhibit EGF-r degradation, (iii) inhibition of EGF induced EGF-r degradation, (iv) protect threonine phosphorylation of EGF-r, (v) protect threonine phosphorylation of other molecules, particularly a 62 KD molecule identified by immunoprecipitation, and (vi) inhibit vascular endothelial cell growth factor signal by tumor cells by greater than 50% and endothelial cells by greater than 40% relative to control.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a continuation-in-part application ofU.S. patent application Ser. No. 09/162,280, filed Sep. 28, 1998, whichis a continuation-in-part application of U.S. patent application Ser.No. 08/851,362, filed May 5, 1997.

BACKGROUND OF THE INVENTION

[0002] 1. Summary of the Invention

[0003] In accordance with the present invention, there are providedfilly human contiguous heavy and light chain sequences spanning thecomplementarity determining regions monoclonal antibodies against humanepidermal growth factor receptor (EGF-r). Nucelotide sequences encodingand amino acid sequences comprising heavy and light chain immunoglobulinmolecules, particularly sequences corresponding to (CDR's), specificallyfrom CDR1 through CDR3, are provided. Hybridomas expressing suchimmunoglobulin molecules and monoclonal antibodies are also provided.Also provided in accordance with the invention are antibodies thatpossess one or more of the following functional characteristics: (i)inhibit tyrosine phosphorylation of EGF-r, (ii) do not inhibit EGF-rinternalization, (ii) inhibit EGF-r degradation, (iii) inhibition of EGFinduced EGF-r degradation, (iv) protect threonine phosphorylation ofEGF-r, (v) protect threonine phosphorylation of other molecules,particularly a 62 KD molecule identified by immunoprecipitation, and(vi) inhibit vascular endothelial cell growth factor signal by tumorcells by greater than 50% and endothelial cells by greater than 40%relative to control.

[0004] 2. Background of the Technology

[0005] Most applications of monoclonal antibodies (MAbs) in cancertherapy rely on the ability of the antibody to specifically deliver tothe cancerous tissues cytotoxic effector functions such asimmune-enhancing isotypes, toxins or drugs. An alternative approach isto utilize MAbs to directly affect the survival of tumor cells bydepriving them of essential extracellular proliferation signals, such asthose mediated by growth factors through their cell receptors. One ofthe attractive targets in this approach is the epidermal growth factorreceptor (EGFr), which binds EGF and transforming growth factor α(TGFα)(1-4). Binding of EGF or TGFα to EGFr, a 170 kDa transmembrane ellsurface glycoprotein, triggers a cascade of cellular biochemical events,including EGFr autophosphorylation and internalization, which culminatesin cell proliferation (1).

[0006] Several observations implicate EGFr in supporting development andprogression of human solid tumors. Overexpression of EGFr has been shownto induce transformed properties in recipient cells (5). EGFr expressionhas been found to be up-regulated on many human tumors, including lung,colon, breast, prostate, brain, head and neck, ovarian and renalcarcinoma, and the increase in receptor levels has been reported to beassociated with a poor clinical prognosis (2, 3, 6-8). In many cases,the increased surface EGFr expression was accompanied by production ofTGFα or EGF by the tumor cells, suggesting the involvement of anautocrine growth control in the progression of these tumors (2, 3, 6,8). These observations suggested that blocking the interaction betweenthe growth factors and EGFr could result in arrest of tumor growth andpossibly affect tumor survival (2-4,6).

[0007] MAbs specific to the human EGFr, capable of neutralizing EGF andTGFα binding to tumor cells and of inhibiting ligand-mediated cellproliferation in vitro, have been generated from mice and rats(2,3,4,6). Some of these antibodies, such as the mouse 108 (9) 225 and528 (2,3) or the rat ICR16, ICR62 and ICR64 (6,10, 11) MAbs, wereevaluated extensively for their ability to affect tumor growth inxenograft mouse models. Most of the anti-EGFr MAbs were efficacious inpreventing tumor formation in athymic mice when administered togetherwith the human tumor cells (2,11). When injected into mice bearingestablished human tumor xenografts, the mouse MAbs 225 and 528 causedpartial tumor regression and required the co-administration ofchemotherapeutic agents, such as doxorubicin or cisplatin, foreradication of the tumors (2, 3,12, 13). A chimeric version of the 225MAb (C225), in which the mouse antibody variable regions are linked tohuman constant regions, exhibited an improved in vivo anti-tumoractivity but only at high doses (14, 15). The rat ICR16, ICR62, andICR64 antibodies caused regression of established tumors but not theircomplete eradication (11). These results established EGFr as a promisingtarget for antibody therapy against EGFr-expressing solid tumors and ledto human clinical trials with the C225 MAb in multiple human solidcancers (2,3,6). However, the limited efficacy of these MAbs asmonotherapeutic agents required their assessment in combination withchemotherapy (16, 17). This requirement can limit the utilization ofanti-EGFr antibodies in patients for whom chemotherapy is not available.Therefore, the identification of an anti-EGFr antibody capable oferadicating established human tumors by itself can expand the patientpopulations and cancer indications to which EGFr antibody therapy can beapplied successfully. In addition, the MAbs currently pursued in humanclinical trials, being murine chimeric antibodies (2,4), are likely toinduce immunogenic or allergic responses to the mouse components inimmunocompetent patients, leading to reduction in the antibody efficacyand safety. Therefore, anti-EGFr antibody therapy can be fully evaluatedwith the availability of a fully human anti-EGFr antibody that exhibitstherapeutic efficacy on EGFr-expressing tumors and that can beadministered repeatedly to all appropriate patient populations.

[0008] EGF-r has been demonstrated to be overexpressed on many types ofhuman solid tumors. Mendelsohn Cancer Cells 7:359 (1989), MendelsohnCancer Biology 1:339-344 (1990), Modjtahedi and Dean Int'l J. Oncology4:277-296 (1994). For example, EGF-r overexpression has been observed incertain lung, breast, colon, gastric, brain, bladder, head and neck,ovarian, and prostate carcinomas. Modjtahedi and Dean Int'l J. Oncology4:277-296 (1994). Both epidermal growth factor (EGF) and transforminggrowth factor-alpha (TGF-α) have been demonstrated to bind to EGF-r andto lead to cellular proliferation and tumor growth.

[0009] Thus, certain groups have proposed that antibodies against EGF,TGF-α, and EGF-r may be useful in the therapy of tumors expressing oroverexpressing EGF-r. Mendelsohn Cancer Cells 7:359 (1989), MendelsohnCancer Biology 1:339-344 (1990), Modjtahedi and Dean Int'l J. Oncology4:277-296 (1994), Tosi et al. Int'l J. Cancer 62:643-650 (1995). Indeed,it has been demonstrated that anti-EGF-r antibodies while blocking EGFand TGF-α binding to the receptor appear to inhibit tumor cellproliferation. At the same time, however, anti-EGF-r antibodies have notappeared to inhibit EGF and TGF-α independent cell growth. Modjtahediand Dean Int'l J. Oncology 4:277-296 (1994).

[0010] In view of these findings, a number of murine and rat monoclonalantibodies against EGF-r have been developed and tested for theirability inhibit the growth of tumor cells in vitro and in vivo.Modjtahedi and Dean Int'l J. Oncology 4:277-296 (1994). The antibodythat has apparently advanced the farthest in the clinic is a chimericantibody, designated C225, which has a murine variable region and ahuman IgGI constant region. Modjtahedi and Dean Int'l J Oncology4:277-296 (1994). The murine antibody, designated 225, upon which theC225 antibody is based, was developed by University of California andRorer. See U.S. Pat. No. 4,943,533 and European Patent No. 359,282, thedisclosures of which are hereby incorporated by reference. The C225antibody was demonstrated to inhibit EGF-mediated tumor cell growth invitro and inhibit human tumor formation in vivo in nude mice. Theantibody, moreover, appeared to act in synergy with certainchemotherapeutic agents to eradicate human tumors in vivo in xenograftmouse models. Modjtahedi and Dean Int'l J. Oncology 4:277-296 (1994).

[0011] ImClone has been conducting human clinical trials using theanti-EGF-r antibody designated C225. Phase I and Phase I/II clinicaltrials in patients with head and neck, prostate, and lung carcinomasapparently have been, or are currently being, conducted with C225. InPhase I clinical trials, no toxicity was detected with multipleinjections and with doses of up to perhaps 400 mg/m², even in casesinvolving immuno compromised patients. Such studies were conducted asdose escalation studies comprising 5 doses of from about 5 to about 200mg/m² and were performed in combination with chemotherapy (i.e.,doxorubicin, adriamycin, taxol, and cisplatin). In addition to theapparent safety data that has been generated in these studies,preliminary results from the studies appear to indicate some evidence oftumor shrinkage in 80% of patients having prostate cancer.

[0012] Each of these above-mentioned antibodies, however, possess murineor rat variable and/or constant regions. The presence of such murine orrat derived proteins can lead to the rapid clearance of the antibodiesor can lead to the generation of an immune response against the antibodyby a patient. In order to avoid the utilization of murine or rat derivedantibodies, it has been postulated that one could introduce humanantibody function into a rodent so that the rodent would produce fullyhuman antibodies.

[0013] The ability to clone and reconstruct megabase-sized human loci inYACs and to introduce them into the mouse germline provides a powerfulapproach to elucidating the functional components of very large orcrudely mapped loci as well as generating useful models of humandisease. Furthermore, the utilization of such technology forsubstitution of mouse loci with their human equivalents could provideunique insights into the expression and regulation of human geneproducts during development, their communication with other systems, andtheir involvement in disease induction and progression.

[0014] An important practical application of such a strategy is the“humanization” of the mouse humoral immune system. Introduction of humanimmunoglobulin (Ig) loci into mice in which the endogenous Ig genes havebeen inactivated offers the opportunity to study the mechanismsunderlying programmed expression and assembly of antibodies as well astheir role in B-cell development. Furthermore, such a strategy couldprovide an ideal source for production of fully human monoclonalantibodies (Mabs)—an important milestone towards fulfilling the promiseof antibody therapy in human disease. Fully human antibodies areexpected to minimize the immunogenic and allergic responses intrinsic tomouse or mouse-derivatized Mabs and thus to increase the efficacy andsafety of the administered antibodies. The use of fully human antibodiescan be expected to provide a substantial advantage in the treatment ofchronic and recurring human diseases, such as inflammation,autoimmunity, and cancer, which require repeated antibodyadministrations.

[0015] One approach towards this goal was to engineer mouse strainsdeficient in mouse antibody production with large fragments of the humanIg loci in anticipation that such mice would produce a large repertoireof human antibodies in the absence of mouse antibodies. Large human Igfragments would preserve the large variable gene diversity as well asthe proper regulation of antibody production and expression. Byexploiting the mouse machinery for antibody diversification andselection and the lack of immunological tolerance to human proteins, thereproduced human antibody repertoire in these mouse strains should yieldhigh affinity antibodies against any antigen of interest, includinghuman antigens. Using the hybridoma technology, antigen-specific humanMabs with the desired specificity could be readily produced andselected.

[0016] This general strategy was demonstrated in connection with ourgeneration of the first XenoMouse™ strains as published in 1994. SeeGreen et al. Nature Genetics 7:13-21 (1994). The XenoMouse™ strains wereengineered with yeast artificial chromosomes (YACs) containing 245 kband 190 kb-sized germline configuration fragments of the human heavychain locus and kappa light chain locus, respectively, which containedcore variable and constant region sequences. Id. The human Ig containingYACs proved to be compatible with the mouse system for bothrearrangement and expression of antibodies and were capable ofsubstituting for the inactivated mouse Ig genes. This was demonstratedby their ability to induce B-cell development, to produce an adult-likehuman repertoire of fully human antibodies, and to generateantigen-specific human Mabs. These results also suggested thatintroduction of larger portions of the human Ig loci containing greaternumbers of V genes, additional regulatory elements, and human Igconstant regions might recapitulate substantially the full repertoirethat is characteristic of the human humoral response to infection andimmunization. The work of Green et al. was recently extended to theintroduction of greater than approximately 80% of the human antibodyrepertoire through introduction of megabase sized, germlineconfiguration YAC fragments of the human heavy chain loci and kappalight chain loci, respectively. See Mendez et al. Nature Genetics15:146-156 (1997) and U.S. patent application Ser. No. 08/759,620, filedDec. 3, 1996, the disclosures of which are hereby incorporated byreference.

[0017] Such approach is further discussed and delineated in U.S. PatentApplication Ser. No. 07/466,008, filed Jan. 12, 1990, Ser. No.07/610,515, filed Nov. 8, 1990, Ser. No. 07/919,297, filed Jul. 24,1992, Ser. No. 07/922,649, filed Jul. 30, 1992, filed 08/031,801, filedMar. 15, 1993, Ser. No. 08/112,848, filed Aug. 27, 1993, Ser. No.08/234,145, filed Apr. 28, 1994, Ser. No. 08/376,279, filed Jan. 20,1995, Ser. No. 08/430, 938, Apr. 27, 1995, Ser. No. 08/464,584, filedJun. 5, 1995, Ser. No. 08/464,582, filed Jun. 5, 1995, Ser. No.08/463,191, filed Jun. 5, 1995, Ser. No. 08/462,837, filed Jun. 5, 1995,Ser. No. 08/486,853, filed Jun. 5, 1995, Ser. No. 08/486,857, filed Jun.5, 1995, Ser. No. 08/486,859, filed Jun. 5, 1995, Ser. No. 08/462,513,filed Jun. 5, 1995, Ser. No. 08/724,752, filed Oct. 2, 1996, and Ser.No. 08/759,620, filed Dec. 3, 1996. See also Mendez et al. NatureGenetics 15:146-156 (1997). See also European Patent No., EP 0 463 151B1, grant published Jun. 12, 1996, International Patent Application No.,WO 94/02602, published Feb. 3, 1994, International Patent ApplicationNo., WO 96/34096, published Oct. 31, 1996, and PCT Application No.PCT/US96/05928, filed Apr. 29, 1996. The disclosures of each of theabove-cited patents, applications, and references are herebyincorporated by reference in their entirety.

[0018] In an alternative approach, others, including GenPharmInternational, Inc., have utilized a “minilocus” approach. In theminilocus approach, an exogenous Ig locus is mimicked through theinclusion of pieces (individual genes) from the Ig locus. Thus, one ormore V_(H) genes, one or more D_(H) genes, one or more J_(H) genes, a muconstant region, and a second constant region (preferably a gammaconstant region) are formed into a construct for insertion into ananimal. This approach is described in U.S. Pat. No. 5,545,807 to Suraniet al. and U.S. Pat. Nos. 5,545,806 and 5,625,825, both to Lonberg andKay, and GenPharm International U.S. Patent Application Ser. No.07/574,748, filed Aug. 29, 1990, Ser. No. 07/575,962, filed Aug. 31,1990, Ser. No. 07/810,279, filed Dec. 17, 1991, Ser. No. 07/853,408,filed Mar. 18, 1992, Ser. No. 07/904,068, filed Jun. 23, 1992, Ser. No.07/990,860, filed Dec. 16, 1992, Ser. No. 08/053,131, filed Apr. 26,1993, Ser. No. 08/096,762, filed Jul. 22, 1993, Ser. No. 08/155,301,filed Nov. 18, 1993, Ser. No. 08/161,739, filed Dec. 3, 1993, Ser. No.08/165,699, filed Dec. 10, 1993, Ser. No. 08/209,741, filed Mar. 9,1994, the disclosures of which are hereby incorporated by reference. Seealso International Patent Application Nos. WO 94/25585, published Nov.10, 1994, WO 93/12227, published Jun. 24, 1993, WO 92/22645, publishedDec. 23, 1992, WO 92/03918, published Mar. 19, 1992, the disclosures ofwhich are hereby incorporated by reference in their entirety. Seefurther Taylor et al., 1992, Chen et al., 1993, Tuaillon et al., 1993,Choi et al., 1993, Lonberg et al., (1994), Taylor et al., (1994), andTuaillon et al., (1995), the disclosures of which are herebyincorporated by reference in their entirety.

[0019] The inventors of Surani et al., cited above and assigned to theMedical Research Counsel (the “MRC”), produced a transgenic mousepossessing an Ig locus through use of the minilocus approach. Theinventors on the GenPharm International work, cited above, Lonberg andKay, following the lead of the present inventors, proposed inactivationof the endogenous mouse Ig locus coupled with substantial duplication ofthe Surani et al. work.

[0020] An advantage of the minilocus approach is the rapidity with whichconstructs including portions of the Ig locus can be generated andintroduced into animals. Commensurately, however, a significantdisadvantage of the minilocus approach is that, in theory, insufficientdiversity is introduced through the inclusion of small numbers of V, D,and J genes. Indeed, the published work appears to support this concern.B-cell development and antibody production of animals produced throughuse of the minilocus approach appear stunted. Therefore, researchsurrounding the present invention has consistently been directed towardsthe introduction of large portions of the Ig locus in order to achievegreater diversity and in an effort to reconstitute the immune repertoireof the animals.

[0021] Human anti-mouse antibody (HAMA) responses have led the industryto prepare chimeric or otherwise humanized antibodies. While the C225antibody is a chimeric antibody, having a human constant region and amurine variable region, it is expected that certain human anti-chimericantibody (HACA) responses will be observed, particularly in chronic ormulti-dose utilizations of the antibody.

[0022] Thus, it would be desirable to provide fully human antibodiesagainst EGF-r that possess similar or enhanced activities as compared toC225 in order to vitiate concerns and/or effects of HAMA or HACAresponse.

BRIEF DESCRIPTION OF THE DRAWING FIGURES

[0023]FIG. 1 is an amino acid sequence of a heavy chain immunoglobulinmolecule that is secreted by the hybridoma E10.1. Differences betweenthe sequence encoded by heavy chain variable gene 4-31 and the sequenceof the E1.1 secreted heavy chain are indicated in bold and enlargedfont. The contiguous sequence from CDR1 through CDR3 is indicated byunderlining and CDR1, CDR2, and CDR3 sequences are each indicated bydouble underlining.

[0024]FIG. 2 is a nucleotide sequence of the cDNA encoding the heavychain immunoglobulin molecule of FIG. 1 that was cloned out of thehybridoma E1.1.

[0025]FIG. 3 is an amino acid sequence of a kappa light chainimmunoglobulin molecule that is secreted by the hybridoma E1.1.Differences between the sequence encoded by light chain variable gene018 and the sequence of the E1.1 secreted light chain are indicated inbold and enlarged font. The contiguous sequence from CDR1 through CDR3is indicated by underlining and CDR1, CDR2, and CDR3 sequences are eachindicated by double underlining.

[0026]FIG. 4 is a nucleotide sequence of the cDNA encoding the kappalight chain immunoglobulin molecule of FIG. 3 that was cloned out of thehybridoma E1.1.

[0027]FIG. 5 is an amino acid sequence of a heavy chain immunoglobulinmolecule that is secreted by the hybridoma E2.4. Differences between thesequence encoded by heavy chain variable gene 4-31 and the sequence ofthe E2.4 secreted heavy chain are indicated in bold and enlarged font.The contiguous sequence from CDR1 through CDR3 is indicated byunderlining and CDR1, CDR2, and CDR3 sequences are each indicated bydouble underlining.

[0028]FIG. 6 is a nucleotide sequence of the cDNA encoding the heavychain immunoglobulin molecule of FIG. 5 that was cloned out of thehybridoma E2.4.

[0029]FIG. 7 is an amino acid sequence of a kappa light chainimmunoglobulin molecule that is secreted by the hybridoma E2.4.Differences between the sequence encoded by light chain variable gene018 and the sequence of the E2.4 secreted light chain are indicated inbold and enlarged font. The contiguous sequence from CDR1 through CDR3is indicated by underlining and CDR1, CDR2, and CDR3 sequences are eachindicated by double underlining.

[0030]FIG. 8 is a nucleotide sequence of the cDNA encoding the kappalight chain immunoglobulin molecule of FIG. 7 that was cloned out of thehybridoma E2.4.

[0031]FIG. 9 is an amino acid sequence of a heavy chain immunoglobulinmolecule that is secreted by the hybridoma E2.5. Differences between thesequence encoded by heavy chain variable gene 4-31 and the sequence ofthe E2.5 secreted heavy chain are indicated in bold and enlarged font.The contiguous sequence from CDR1 through CDR3 is indicated byunderlining and CDR1, CDR2, and CDR3 sequences are each indicated bydouble underlining.

[0032]FIG. 10 is a nucleotide sequence of the cDNA encoding the heavychain immunoglobulin molecule of FIG. 9 that was cloned out of thehybridoma E2.5.

[0033]FIG. 11 is an amino acid sequence of a kappa light chainimmunoglobulin molecule that is secreted by the hybridoma E2.5.Differences between the sequence encoded by light chain variable gene018 and the sequence of the E2.5 secreted light chain are indicated inbold and enlarged font. The contiguous sequence from CDR1 through CDR3is indicated by underlining and CDR1, CDR2, and CDR3 sequences are eachindicated by double underlining.

[0034]FIG. 12 is a nucleotide sequence of the cDNA encoding the kappalight chain immunoglobulin molecule of FIG. 11 that was cloned out ofthe hybridoma E2.5.

[0035]FIG. 13 is an amino acid sequence of a heavy chain immunoglobulinmolecule that is secreted by the hybridoma E6.2. Differences between thesequence encoded by heavy chain variable gene 4-31 and the sequence ofthe E6.2 secreted heavy chain are indicated in bold and enlarged font.The contiguous sequence from CDR1 through CDR3 is indicated byunderlining and CDR1, CDR2, and CDR3 sequences are each indicated bydouble underlining.

[0036]FIG. 14 is a nucleotide sequence of the cDNA encoding the heavychain immunoglobulin molecule of FIG. 13 that was cloned out of thehybridoma E6.2.

[0037]FIG. 15 is an amino acid sequence of a kappa light chainimmunoglobulin molecule that is secreted by the hybridoma E6.2.Differences between the sequence encoded by light chain variable gene018 and the sequence of the E6.2 secreted light chain are indicated inbold and enlarged font. The contiguous sequence from CDR1 through CDR3is indicated by underlining and CDR1, CDR2, and CDR3 sequences are eachindicated by double underlining.

[0038]FIG. 16 is a nucleotide sequence of the cDNA encoding the kappalight chain immunoglobulin molecule of FIG. 15 that was cloned out ofthe hybridoma E6.2.

[0039]FIG. 17 is an amino acid sequence of a heavy chain immunoglobulinmolecule that is secreted by the hybridoma E6.4. Differences between thesequence encoded by heavy chain variable gene 4-31 and the sequence ofthe E6.4 secreted heavy chain are indicated in bold and enlarged font.The contiguous sequence from CDR1 through CDR3 is indicated byunderlining and CDR1, CDR2, and CDR3 sequences are each indicated bydouble underlining.

[0040]FIG. 18 is a nucleotide sequence of the CDNA encoding the heavychain immunoglobulin molecule of FIG. 17 that was cloned out of thehybridoma E6.2.

[0041]FIG. 19 is an amino acid sequence of a kappa light chainimmunoglobulin molecule that is secreted by the hybridoma E6.4.Differences between the sequence encoded by light chain variable gene018 and the sequence of the E6.4 secreted light chain are indicated inbold and enlarged font. The contiguous sequence from CDR1 through CDR3is indicated by underlining and CDR1, CDR2, and CDR3 sequences are eachindicated by double underlining.

[0042]FIG. 20 is a nucleotide sequence of the cDNA encoding the kappalight chain immunoglobulin molecule of FIG. 19 that was cloned out ofthe hybridoma E6.4.

[0043]FIG. 21 is an amino acid sequence of a heavy chain immunoglobulinmolecule that is secreted by the hybridoma E2.11. Differences betweenthe sequence encoded by heavy chain variable gene 4-61 and the sequenceof the E2.11 secreted heavy chain are indicated in bold and enlargedfont. The contiguous sequence from CDR1 through CDR3 is indicated byunderlining and CDR1, CDR2, and CDR3 sequences are each indicated bydouble underlining.

[0044]FIG. 22 is a nucleotide sequence of the cDNA encoding the heavychain immunoglobulin molecule of FIG. 21 that was cloned out of thehybridoma E2.11.

[0045]FIG. 23 is an amino acid sequence of a kappa light chainimmunoglobulin molecule that is secreted by the hybridoma E2.11.Differences between the sequence encoded by light chain variable gene018 and the sequence of the E2.11 secreted light chain are indicated inbold and enlarged font. The contiguous sequence from CDR1 through CDR3is indicated by underlining and CDR1, CDR2, and CDR3 sequences are eachindicated by double underlining.

[0046]FIG. 24 is a nucleotide sequence of the cDNA encoding the kappalight chain immunoglobulin molecule of FIG. 23 that was cloned out ofthe hybridoma E2.11.

[0047]FIG. 25 is an amino acid sequence of a heavy chain immunoglobulinmolecule that is secreted by the hybridoma E6.3. Differences between thesequence encoded by heavy chain variable gene 4-61 and the sequence ofthe E6.3 secreted heavy chain are indicated in bold and enlarged font.The contiguous sequence from CDR1 through CDR3 is indicated byunderlining and CDR1, CDR2, and CDR3 sequences are each indicated bydouble underlining.

[0048]FIG. 26 is a nucleotide sequence of the cDNA encoding the heavychain immunoglobulin molecule of FIG. 25 that was cloned out of thehybridoma E6.3.

[0049]FIG. 27 is an amino acid sequence of a kappa light chainimmunoglobulin molecule that is secreted by the hybridoma E6.3.Differences between the sequence encoded by light chain variable gene018 and the sequence of the E6.3 secreted light chain are indicated inbold and enlarged font. The contiguous sequence from CDR1 through CDR3is indicated by underlining and CDR1, CDR2, and CDR3 sequences are eachindicated by double underlining.

[0050]FIG. 28 is a nucleotide sequence of the cDNA encoding the kappalight chain immunoglobulin molecule of FIG. 27 that was cloned out ofthe hybridoma E6.3.

[0051]FIG. 29 is an amino acid sequence of a heavy chain immunoglobulinmolecule that is secreted by the hybridoma E7.6.3. Differences betweenthe sequence encoded by heavy chain variable gene 4-61 and the sequenceof the E7.6.3 secreted heavy chain are indicated in bold and enlargedfont. The contiguous sequence from CDR1 through CDR3 is indicated byunderlining and CDR1, CDR2, and CDR3 sequences are each indicated bydouble underlining.

[0052]FIG. 30 is a nucleotide sequence of the cDNA encoding the heavychain immunoglobulin molecule of FIG. 29 that was cloned out of thehybridoma E7.6.3.

[0053]FIG. 31 is an amino acid sequence of a kappa light chainimmunoglobulin molecule that is secreted by the hybridoma E7.6.3.Differences between the sequence encoded by light chain variable gene018 and the sequence of the E7.6.3 secreted light chain are indicated inbold and enlarged font. The contiguous sequence from CDR1 through CDR3is indicated by underlining and CDR1, CDR2, and CDR3 sequences are eachindicated by double underlining.

[0054]FIG. 32 is a nucleotide sequence of the cDNA encoding the kappalight chain immunoglobulin molecule of FIG. 31 that was cloned out ofthe hybridoma E7.6.3.

[0055]FIG. 33 provides a comparison of specific anti-EGF-r antibodyheavy chain amino acid sequence comparisons with the amino acid sequenceof the particular VH gene which encodes the heavy chain of theparticular antibody.

[0056]FIG. 34 provides a comparison of specific anti-EGF-r antibodylight chain amino acid sequence comparisons with the amino acid sequenceof the particular VK gene which encodes the light chain of theparticular antibody.

[0057]FIG. 35 shows blockage EGF binding to human epidermoid carcinomaA431 cells by human anti-EGF-r antibodies in vitro, where (□) depictsthe results achieved by an anti-EGF-r antibody in accordance with theinvention, () depicts the results achieved by the murine monoclonalantibody 225, and (▴) depicts the results achieved by a control,nonspecific, human IgG2 antibody.

[0058]FIG. 36 shows inhibition of EGF binding to human epidermoidcarcinoma A431 cells by human anti-EGF-r antibodies in vitro, where (□)depicts the results achieved by the murine monoclonal antibody 225, (∘)depicts the results achieved by the murine monoclonal antibody 528, (▾)depicts the results achieved using the E1.1 antibody in accordance withthe invention, (▴) depicts the results achieved using the E2.4 antibodyin accordance with the invention, (

) depicts the results achieved using the E2.5 antibody in accordancewith the invention, (

) depicts the results achieved using the E2.6 antibody in accordancewith the invention, (♦) depicts the results achieved using the E2.11antibody in accordance with the invention, and

(depicts the results achieved using a control, nonspecific human IgG2antibody.

[0059]FIG. 37 shows inhibition of TGF-α binding to human epidermoidcarcinoma A431 cells by human anti-EGF-r antibodies in vitro, where (□)depicts the results achieved by the murine monoclonal antibody 225, (♦)depicts the results achieved using the E6.2 antibody in accordance withthe invention, () depicts the results achieved using the E6.3 antibodyin accordance with the invention, (▴) depicts the results achieved usingthe E7.2 antibody in accordance with the invention, (▪) depicts theresults achieved using the E7.10 antibody in accordance with theinvention, (▾) depicts the results achieved using the E7.6.3, and({circle over (X)}) depicts the results achieved using a control,nonspecific human IgG2 antibody.

[0060]FIG. 38 shows inhibition of EGF binding to human colon carcinomaSW948 cells by human anti-EGF-r antibodies in vitro, where () depictsthe results achieved by an anti-EGF-r antibody in accordance with theinvention, (□) depicts the results achieved by the murine monoclonalantibody 225, and (▴) depicts the results achieved by a control,nonspecific, human IgG2 antibody.

[0061]FIG. 39 shows that human anti-EGF-r antibodies derived fromXenoMouse II strains inhibit growth of SW948 cells in vitro, where (◯)depicts the results achieved by an anti-EGF-r antibody in accordancewith the invention, (□) depicts the results achieved by the murinemonoclonal antibody 225, and (▴) depicts the results achieved by acontrol, nonspecific, human IgG2 antibody.

[0062]FIG. 40 shows the inhibition of human epidermoid carcinoma A431cell growth in nude mice through use of human anti-EGF-r antibodies inaccordance with the invention in vivo. In the Figure, (▴) depicts theresults achieved with a dosage of 1 mg of a human anti-EGF-r antibody inaccordance with the present invention, (V)depicts the results achievedwith a dosage of 0.2 mg of a human anti-EGF-r antibody in accordancewith the present invention, (□) depicts the results achieved by acontrol, nonspecific, human IgG2 antibody, and (◯) depicts the resultsachieved utilizing phosphate buffered saline as a control.

[0063]FIG. 41 shows data related to the inhibition of epidermoidcarcinoma formation in nude mice through use of human anti-EGF-rantibodies in accordance with the invention in vivo showing tumorincidence at day 19.

[0064]FIG. 42 shows data related to the inhibition of epidermoidcarcinoma formation in nude mice through use of human anti-EGF-rantibodies in accordance with the invention in vivo showing tumorincidence at day 120.

[0065]FIG. 43 shows data related to the eradication of an establishedhuman epidermoid tumor in nude mice through use of human anti-EGF-rantibodies in accordance with the invention in vivo. In the Figure, (▴)depicts the results achieved with multiple doses of 1 mg each of a humananti-EGF-r antibody in accordance with the present invention (E7.6.3),(×) depicts the results achieved with two doses of 125 μg each ofdoxorubicin, (*) depicts the results achieved with a multiple doses of 1mg each of a human anti-EGF-r antibody in accordance with the presentinvention (E7.6.3) in combination with two doses of 125 μg each ofdoxorubicin, (▪) depicts the results achieved by a control, nonspecific,human IgG2 antibody, and (♦) depicts the results achieved utilizingphosphate buffered saline as a control.

[0066]FIG. 44 shows data related to the eradication of an establishedhuman epidermoid tumor in nude mice through use of human anti-EGF-rantibodies in accordance with the invention in vivo. In the Figure, (♦)depicts the results achieved with multiple doses of 0.5 mg each of ahuman anti-EGF-r antibody in accordance with the present invention(E2.5), (▪) depicts the results achieved with two doses of 125 μg eachof doxorubicin, (▴) depicts the results achieved with multiple doses of0.5 mg each of a human anti-EGF-r antibody in accordance with thepresent invention (E2.5) in combination with two doses of 125 μg each ofdoxorubicin, (×) depicts the results achieved utilizing phosphatebuffered saline as a control, and (*) depicts the results achievedutilizing a control, nonspecific, human IgG2 antibody at a dose of 1 mg.

[0067]FIG. 45 shows the inhibition of EGF binding to EGFr by anti-EGFrMAbs. The binding of ¹²⁵I-EGF (0.1 nM) to (A) A431 or (B) SW948 cellswas determined in the presence of XenoMouse-derived human (▪ E7.6.3; E2.5.1; ▴ E2.3.1; ∇ E7.5.2; ◯ E7.8.2) or murine (▾ 225; ⋄ 528) anti-EGFrantibodies, or in the presence of the human IgG₂κ control antibody(hIgG₂K). The binding of ¹²⁵I-EGF to the cells in the absence ofantibodies was designated as 100%. The data shown are representative ofmultiple experiments.

[0068]FIG. 46 shows the inhibition of EGF-induced tyrosinephosphorylation of EGFr by E7.6.3 MAb. A431 were incubated with orwithout EGF (16 nM), in the absence or presence of increasingconcentrations of E7.6.3 MAb (0.2-133 nM) as described in “[Materialsand Methods]”. Total EGFr and EGFr tyrosine phosphorylation in celllysates was visualized (A) and quantitated (B) using Western blotanalysis using an anti-phosphotyrosine antibody as described in“Materials and Methods”.

[0069]FIG. 47 shows the Inhibition of EGF-mediated cell activation byanti-EGFr antibodies. A. Activation of A431 cells by 1.67 nM EGF, in theabsence or presence of different concentrations of E7.6.3, was measuredby Cytosensor as changes in extracellular acidification rate. The arrowindicates the times when EGF and/or E7.6.3 were added to the cells. Theresponse is presented as % of baseline acidification rate (designated as100%). B. Effect of increasing concentrations of E7.6.3 and controlPK16.3.1 antibodies on A431 cell activation induced by EGF (1.67 nM), asdetermined by Cytosensor. The response to EGF was measured at the peakacidification rate shown in A. The response in the absence of antibodieswas designated as 100%. The data shown are representative of 2 differentexperiments.

[0070]FIG. 48 shows the inhibition of in vitro tumor cell proliferationby anti-EGFr antibodies. A431 (A) or MDA-468 (B) cells were culturedwith anti-EGFr MAbs (E7.6.3; ♦ 225; ▴ 528) or control human myelomaIgG₂κ (∘), as described in Materials and Methods. Cell viability wasassayed by crystal violet staining. Data presented as % of cell growthinhibition.

[0071]FIG. 49 shows the eradication of established A431 tumor xenograftsby E7.6.3 MAb. A431 cells (5×10⁶) were injected s.c. into the nude miceon day 0. A. At day 7 when tumor size reached an average volume of0.1˜0.25 cm², mice (n=5) were injected i.p. with PBS (∘) or with 1 mg ofeither E7.6.3 (♦) or the control human myeloma IgG₂κ (▪) antibodiestwice a week for three weeks. B. when the mean tumor sizes reached 0.13(▴), 0.56 (▾), 0.73 (♦) or 1.2 () cm³, mice (n=10) were treated with 1mg E7.6.3, twice a week for three weeks. Control mice (∘, n=10) receivedno treatment. C, at day 10 when tumor sizes reached 0.15 cm³, mice (n=8) were injected i.p. with 200 μg (∇) or 50 μg (▴) doses of E7.6.3, or200 μg (▾) or 50 μg () doses of 225 MAbs, twice a week for three weeks.Control mice (∘) received no treatment. Tumors were measured weekly andtheir volume was measured as described in “Materials and Methods”. Thedata is presented as the mean tumor size±SEM.

[0072]FIG. 50 shows the effect of the E7.6.3 Mab on the growth ofestablished human tumor xenografts. 5×10⁶ MDA468 (A) or A431 (B) cellswere injected s.c. into the nude mice on day 0. A. 7 days followinginjection of MDA468 cells, mice (n =8) were injected i.p. with 2 mgE7.6.3 once a week for two weeks (▴). Control mice (n=8) received notreatment (∘). B. Mice (n =10) were given 0.5 mg E7.6.3 via i.p. (▪),i.v. (Δ), s.c. (▾) or i.m. (♦) injections twice a week for three weeks.Control mice (□) received no treatment. The data represents the mean±SEM.

[0073]FIG. 51 shows certain histopathology of E7.6.3-treated A431xenografts. A. Mice with established A431 xenografts were treated i.p.with 0.5 mg E7.6.3 twice a week for three weeks. On day 76 after tumorcells (5×106) injection, tumor-like nodules were excised and examinedhistologically as described in Materials and Methods. B. Histologicalanalysis of A431 tumor xenografts excised from an untreated mouse.

[0074]FIG. 52 is a table the prevention of tumor formation by the E7.6.3MAb. On day 0, mice were injected s.c. with 5×10⁶ A431 cells and i.p.with PBS, 1 mg of control antibody PK16.3.1, 0.2 mg or 1 mg of E7.6.3MAb twice a week, for three weeks. Incidence of tumor formation isexpressed as the number of mice with visible tumors/total number of micewithin each group. ND: not determined.

[0075]FIG. 53 is a table showing the eradication of established A43 1xenograft tumors by E7.6.3 MAb. Nude mice with established A431xenografts (tumor size of 0.13-0.25 cm³ at day 7-10) were treated i.p.with various doses of E7.6.3 MAb or human myeloma IgG₂κ control antibodytwice a week, for three weeks. The table summarizes the results of 11experiments. Mice that received no treatment or control IgG₂κ antibodywere sacrificed between day 35 and 50.

[0076]FIG. 54 is a western blot showing the inhibitory effects of theE7.6.3 antibody on EGF-induced tyrosine phosphoylation and degradationof EGFr in cultured A431 cells.

[0077]FIG. 55 is a western blot showing preliminary results obtainedcomparing the inhibitory effects of the E7.6.3 and 225 antibodies onEGF-induced tyrosine phosphoylation and degradation of EGFr in culturedA431 cells.

[0078]FIG. 56 is a western blot showing preliminary results obtainedcomparing the inhibitory effects of the E7.6.3 and 225 antibodies onEGF-induced tyrosine phosphoylation and degradation of EGFr in culturedA43 1 cells.

[0079]FIG. 57 provides oligonucleotide and amino acid sequenceinformation on the heavy chain of the antibody produced by the E20.1hybridoma.

[0080]FIG. 58 provides oligonucleotide and amino acid sequenceinformation on the light chain of the antibody produced by the E20.1hybridoma.

[0081]FIG. 59 provides oligonucleotide and amino acid sequenceinformation on the heavy chain of the antibody produced by the E20.3hybridoma.

[0082]FIG. 60 provides oligonucleotide and amino acid sequenceinformation on the light chain of the antibody produced by the E20.3hybridoma.

[0083]FIG. 61 provides oligonucleotide and amino acid sequenceinformation on the heavy chain of the antibody produced by the E20.8.1hybridoma.

[0084]FIG. 62 provides oligonucleotide and amino acid sequenceinformation on the light chain of the antibody produced by the E20.8.1hybridoma.

[0085]FIG. 63 provides oligonucleotide and amino acid sequenceinformation on the heavy chain of the antibody produced by the E20.11.2hybridoma.

[0086]FIG. 64 provides oligonucleotide and amino acid sequenceinformation on the light chain of the antibody produced by the E20.11.2hybridoma.

[0087]FIG. 65 provides oligonucleotide and amino acid sequenceinformation on the heavy chain of the antibody produced by the E20.18hybridoma.

[0088]FIG. 66 provides oligonucleotide and amino acid sequenceinformation on the light chain of the antibody produced by the E20.18hybridoma.

[0089]FIG. 67 provides oligonucleotide and amino acid sequenceinformation on the heavy chain of the antibody produced by the E20.19.2hybridoma.

[0090]FIG. 68 provides oligonucleotide and amino acid sequenceinformation on the light chain of the antibody produced by the E20.19.2hybridoma.

[0091]FIG. 69 provides oligonucleotide and amino acid sequenceinformation on the heavy chain of the antibody produced by the E20.21hybridoma.

[0092]FIG. 70 provides oligonucleotide and amino acid sequenceinformation on the heavy chain of the antibody produced by the E20.22hybridoma.

[0093]FIG. 71 provides a mutation analysis of antibodies in accordancewith the invention. In particular, the sequence of the E20.21 antibody,which comprises a VH 4-31 heavy chain is shown.

[0094]FIG. 72 provides oligonucleotide and amino acid sequenceinformation on the heavy chain of the antibody produced by the E7.5.2hybridoma.

[0095]FIG. 73 provides oligonucleotide and amino acid sequenceinformation on the light chain of the antibody produced by the E7.5.2hybridoma.

[0096]FIG. 74 shows data related to the eradication of an establishedhuman epidermoid tumor in nude mice through use of human anti-EGF-rneutralizing antibody E7.6.3 in accordance with the invention in vivo.A431 cells (5×10⁶) were injected s.c. into the nude mice on day 0. Atday 8 when tumor become established, mice (n=10) were injected i.p. with1 mg of either E7.6.3 (filled square) or E7.5.2 (filled triangle), orreceived no treatment as a control (open circle) twice a week for threeweeks. The arrows indicate the timing and number of antibody injections.Tumors were measured twice a week and their volume was measured asdescribed in the “Materials and Methods” section of Example 14. The datais presented as the mean tumor size±SEM.

[0097]FIG. 75 shows the effect of the E7.6.3 Mab on the growth ofestablished human pancreatic tumor xenografts. HPAC cells (5×10⁶) wereinjected s.c. into the nude mice on day 0. At day 7 when tumor becomeestablished, mice (n=10) were injected i.p. with 1 mg of E7.6.3 (filledsquare) or received no treatment as a control (open circle) twice a weekfor three weeks. The arrows indicate the timing and number of antibodyinjections. Tumors were measured twice a week and their volume wasmeasured as described in the “Materials and Methods” section of Example14. The data is presented as the mean tumor size±SEM.

[0098]FIG. 76 shows the inhibition of the growth of established humanpancreatic tumor xenografts. BxPC3 cells (5×10⁶) were injected s.c. intothe nude mice on day 0. At day 7 when tumor become established, mice(n=10) were injected i.p. with 1 mg of E7.6.3 (filled square) orreceived no treatment as a control (open circle) twice a week for threeweeks. The arrows indicate the timing and number of antibody injections.Tumors were measured twice a week and their volume was measured asdescribed in the “Materials and Methods” section of Example 14. The datais presented as the mean tumor size±SEM.

[0099]FIG. 77 shows the inhibition of the growth of established humanpancreatic tumor xenografts. Hs77T9 cells (5×10⁶) were injected s.c.into the nude mice on day 0. At day 7 when tumor become established,mice (n=10) were injected i.p. with 1 mg of E7.6.3 (filled square) orreceived no treatment as a control (open circle) twice a week for threeweeks. The arrows indicate the timing and number of antibody injections.Tumors were measured twice a week and their volume was measured asdescribed in the “Materials and Methods” section of Example 14. The datais presented as the mean tumor size±SEM.

[0100]FIG. 78 shows the inhibition of the growth of established humanrenal tumor xenografts. Sk-RC-29 cells (5×10⁶) were injected s.c. intothe nude mice on day 0. At day 7 when tumor become established, mice(n=10) were injected i.p. with 1 mg of E7.6.3 (filled square) orreceived no treatment as a control (open circle) twice a week for threeweeks. The arrows indicate the timing and number of antibody injections.Tumors were measured twice a week and their volume was measured asdescribed in the “Materials and Methods” section of Example 14. The datais presented as the mean tumor size±SEM.

[0101]FIG. 79 shows the effect of the E7.6.3 Mab on the growth ofestablished human colon tumor xenografts. SW707 (EGF-r⁻) cells (5×10⁶)were injected s.c. into the nude mice on day 0. At day 7 when tumorbecome established, mice (n=10) were injected i.p. with 1 mg of E7.6.3(filled square) or received no treatment as a control (open circle)twice a week for three weeks. The arrows indicate the timing and numberof antibody injections. Tumors were measured twice a week and theirvolume was measured as described in the “Materials and Methods” sectionof Example 14. The data is presented as the mean tumor size±SEM.

[0102]FIG. 80 provides a series of graphs showing the internalization ofEGF-r with panel A showing the internalization of EGF-r based on ¹²⁵I-EGF and panel B showing the internalization of EGF-r based on¹²⁵I-E7.6.3.

[0103]FIG. 81 provides a bar graph that demonstrates the competitiveeffects of antibodies with EGF as a positive control (panel A) for thebar graph in panel B that demonstrates that E7.6.3 is not degraded.

[0104]FIG. 82 is a series of immunoprecipitation blots comparing theeffects of antibodies on EGF-r degradation.

[0105]FIG. 83 is an immunoprecipitation blot comparing the effects ofantibodies on EGF-r threonine phosphorylation.

[0106]FIG. 84 is a western blot comparing the effects of antibodies onother threonine phosphorylation.

[0107]FIG. 85 is a series of bar graphs showing the effects ofantibodies on the production of vascular endothelial cell growth factorin tumor (A431) cells.

[0108]FIG. 86 is a graph showing the effects of antibodies on theproduction of vascular endothelial cell growth factor in endothelial(ECV304) cells.

SUMMARY OF THE INVENTION

[0109] In accordance with the present invention, there is provided anantibody that binds to epidermal growth factor receptor that possessesone or more of the following functional characteristics: (i) inhibittyrosine phosphorylation of EGF-r, (ii) do not inhibit EGF-rinternalization, (ii) inhibit EGF-r degradation, (iii) inhibition of EGFinduced EGF-r degradation, (iv) protect threonine phosphorylation ofEGF-r, (v) protect threonine phosphorylation of other molecules,particularly a 62 KD molecule identified by immunoprecipitation, and(vi) inhibit vascular endothelial cell growth factor signal by tumorcells by greater than 50% and endothelial cells by greater than 40%relative to control.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0110] In accordance with the present invention, there are providedfully human monoclonal antibodies against human epidermal growth factorreceptor (EGF-r). Nucleotide sequences encoding and amino acid sequencescomprising heavy and light chain immunoglobulin molecules, particularlysequences corresponding to a contiguous heavy and light chain sequencesfrom CDR1 through CDR3, are provided. Hybridomas expressing suchimmunoglobulin molecules and monoclonal antibodies are also provided.

[0111] To this end we utilized our human antibody-producing XenoMousestrains to generate potent fully human anti-EGFr MAbs. As previouslydescribed, these mouse strains were engineered to be deficient in mouseantibody production and to contain integrated megabase-sized fragmentsfrom the human heavy and kappa (K) light chain loci with the majority ofthe human antibody gene repertoire (18). The human Ig loci provided theXenoMouse strains with the ability to produce high affinity human MAbsto a broad spectrum of antigens, including human antigens (18, 19). Aspresented in this report, using XenoMouse strains we generated a panelof anti-EGFr fully human IgG₂κ MAbs from which we selected the E7.6.3antibody. This antibody exhibits high affinity (5×10⁻¹ M) to thereceptor, neutralizes both EGF and TGFα binding to EGFr-expressing humancarcinoma cell lines, and inhibits ligand-induced tumor cellproliferation. The antibody not only prevents human tumor formation inathymic mice but, more importantly, effectively eradicates largeestablished human tumor xenografts, independent of chemotherapeuticagents. The potent anti-tumor activity of the E7.6.3 MAb indicates it isa good candidate for use as a monotherapeutic agent for the treatment ofEGFr-expressing human solid tumors.

[0112] Definitions

[0113] Unless otherwise defined, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures utilized in connection with, and techniques of, cell andtissue culture, molecular biology, and protein and oligo- orpolynucleotide chemistry and hybridization described herein are thosewell known and commonly used in the art. Standard techniques are usedfor recombinant DNA, oligonucleotide synthesis, and tissue culture andtransformation (e.g., electroporation, lipofection). Enzymatic reactionsand purification techniques are performed according to manufacturer'sspecifications or as commonly accomplished in the art or as describedherein. The foregoing techniques and procedures are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification. See e.g., Sambrook etal. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989)), which isincorporated herein by reference. The nomenclatures utilized inconnection with, and the laboratory procedures and techniques of,analytical chemistry, synthetic organic chemistry, and medicinal andpharmaceutical chemistry described herein are those well known andcommonly used in the art. Standard techniques are used for chemicalsyntheses, chemical analyses, pharmaceutical preparation, formulation,and delivery, and treatment of patients.

[0114] As utilized in accordance with the present disclosure, thefollowing terms, unless otherwise indicated, shall be understood to havethe following meanings:

[0115] The term “isolated polynucleotide” as used herein shall mean apolynucleotide of genomic, cDNA, or synthetic origin or some combinationthereof, which by virtue of its origin the “isolated polynucleotide” (1)is not associated with all or a portion of a polynucleotide in which the“isolated polynucleotide” is found in nature, (2) is operably linked toa polynucleotide which it is not linked to in nature, or (3) does notoccur in nature as part of a larger sequence.

[0116] The term “isolated protein” referred to herein means a protein ofcDNA, recombinant RNA, or synthetic origin or some combination thereof,which by virtue of its origin, or source of derivation, the “isolatedprotein” (1) is not associated with proteins found in nature, (2) isfree of other proteins from the same source, e.g. free of murineproteins, (3) is expressed by a cell from a different species, or (4)does not occur in nature.

[0117] The term “polypeptide” is used herein as a generic term to referto native protein, fragments, or analogs of a polypeptide sequence.Hence, native protein, fragments, and analogs are species of thepolypeptide genus. Preferred polypeptides in accordance with theinvention comprise the human heavy chain immunoglobulin moleculesrepresented by FIGS. 1, 5, 9, 13, 17, 21, 25, and 29 and the human kappalight chain immunoglobulin molecules represented by FIGS. 3, 7, 11, 15,19, 23, 27, and 31, as well as antibody molecules formed by combinationscomprising the heavy chain immunoglobulin molecules with light chainimmunoglobulin molecules, such as the kappa light chain immunoglobulinmolecules, and vice versa, as well as fragments and analogs thereof.

[0118] The term “naturally-occurring” as used herein as applied to anobject refers to the fact that an object can be found in nature. Forexample, a polypeptide or polynucleotide sequence that is present in anorganism (including viruses) that can be isolated from a source innature and which has not been intentionally modified by man in thelaboratory or otherwise is naturally-occurring.

[0119] The term “operably linked” as used herein refers to positions ofcomponents so described are in a relationship permitting them tofunction in their intended manner. A control sequence “operably linked”to a coding sequence is ligated in such a way that expression of thecoding sequence is achieved under conditions compatible with the controlsequences.

[0120] The term “control sequence” as used herein refers topolynucleotide sequences which are necessary to effect the expressionand processing of coding sequences to which they are ligated. The natureof such control sequences differs depending upon the host organism; inprokaryotes, such control sequences generally include promoter,ribosomal binding site, and transcription termination sequence; ineukaryotes, generally, such control sequences include promoters andtranscription termination sequence. The term “control sequences” isintended to include, at a minimum, all components whose presence isessential for expression and processing, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences.

[0121] The term “polynucleotide” as referred to herein means a polymericform of nucleotides of at least 10 bases in length, eitherribonucleotides or deoxynucleotides or a modified form of either type ofnucleotide. The term includes single and double stranded forms of DNA.

[0122] The term “oligonucleotide” referred to herein includes naturallyoccurring, and modified nucleotides linked together by naturallyoccurring, and non-naturally occurring oligonucleotide linkages.Oligonucleotides are a polynucleotide subset generally comprising alength of 200 bases or fewer. Preferably oligonucleotides are 10 to 60bases in length and most preferably 12, 13, 14, 15, 16, 17, 18, 19, or20 to 40 bases in length. Oligonucleotides are usually single stranded,e.g. for probes; although oligonucleotides may be double stranded, e.g.for use in the construction of a gene mutant. Oligonucleotides of theinvention can be either sense or antisense oligonucleotides.

[0123] The term “naturally occurring nucleotides” referred to hereinincludes deoxyribonucleotides and ribonucleotides. The term “modifiednucleotides” referred to herein includes nucleotides with modified orsubstituted sugar groups and the like. The term “oligonucleotidelinkages” referred to herein includes oligonucleotides linkages such asphosphorothioate, phosphorodithioate, phosphoroselenoate,phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate,phosphoroamidate, and the like. See e.g., LaPlanche et al. Nucl. AcidsRes. 14:9081 (1986); Stec et al. J. Am. Chem. Soc. 106:6077 (1984);Stein et al. Nucl. Acids Res. 16:3209 (1988); Zon et al. Anti-CancerDrug Design 6:539 (1991); Zon et al. Oligonucleotides and Analogues: APractical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford UniversityPress, Oxford England (1991)); Stec et al. U.S. Pat. No. 5,151,510;Uhlmann and Peyman Chemical Reviews 90:543 (1990), the disclosures ofwhich are hereby incorporated by reference. An oligonucleotide caninclude a label for detection, if desired.

[0124] The term “selectively hybridize” referred to herein means todetectably and specifically bind. Polynucleotides, oligonucleotides andfragments thereof in accordance with the invention selectively hybridizeto nucleic acid strands under hybridization and wash conditions thatminimize appreciable amounts of detectable binding to nonspecificnucleic acids. High stringency conditions can be used to achieveselective hybridization conditions as known in the art and discussedherein. Generally, the nucleic acid sequence homology between thepolynucleotides, oligonucleotides, and fragments of the invention and anucleic acid sequence of interest will be at least 80%, and moretypically with preferably increasing homologies of at least 85%, 90%,95%, 99%, and 100%. Two amino acid sequences are homologous if there isa partial or complete identity between their sequences. For example, 85%homology means that 85% of the amino acids are identical when the twosequences are aligned for maximum matching. Gaps (in either of the twosequences being matched) are allowed in maximizing matching; gap lengthsof 5 or less are preferred with 2 or less being more preferred.Alternatively and preferably, two protein sequences (or polypeptidesequences derived from them of at least 30 amino acids in length) arehomologous, as this term is used herein, if they have an alignment scoreof at more than 5 (in standard deviation units) using the program ALIGNwith the mutation data matrix and a gap penalty of 6 or greater. SeeDayhoff, M. O., in Atlas of Protein Sequence and Structure, pp. 101-110(Volume 5, National Biomedical Research Foundation (1972)) andSupplement 2 to this volume, pp. 1-10. The two sequences or partsthereof are more preferably homologous if their amino acids are greaterthan or equal to 50% identical when optimally aligned using the ALIGNprogram. The term “corresponds to” is used herein to mean that apolynucleotide sequence is homologous (i.e., is identical, not strictlyevolutionarily related) to all or a portion of a referencepolynucleotide sequence, or that a polypeptide sequence is identical toa reference polypeptide sequence. In contradistinction, the term“complementary to” is used herein to mean that the complementarysequence is homologous to all or a portion of a reference polynucleotidesequence. For illustration, the nucleotide sequence “TATAC” correspondsto a reference sequence “TATAC” and is complementary to a referencesequence “GTATA”.

[0125] The following terms are used to describe the sequencerelationships between two or more polynucleotide or amino acidsequences: “reference sequence”, “comparison window”, “sequenceidentity”, “percentage of sequence identity”, and “substantialidentity”. A “reference sequence” is a defined sequence used as a basisfor a sequence comparison; a reference sequence may be a subset of alarger sequence, for example, as a segment of a full-length cDNA or genesequence given in a sequence listing or may comprise a complete cDNA orgene sequence. Generally, a reference sequence is at least 18nucleotides or 6 amino acids in length, frequently at least 24nucleotides or 8 amino acids in length, and often at least 48nucleotides or 16 amino acids in length. Since two polynucleotides oramino acid sequences may each (1) comprise a sequence (i.e., a portionof the complete polynucleotide or amino acid sequence) that is similarbetween the two molecules, and (2) may further comprise a sequence thatis divergent between the two polynucleotides or amino acid sequences,sequence comparisons between two (or more) molecules are typicallyperformed by comparing sequences of the two molecules over a “comparisonwindow” to identify and compare local regions of sequence similarity. A“comparison window”, as used herein, refers to a conceptual segment ofat least 18 contiguous nucleotide positions or 6 amino acids wherein apolynucleotide sequence or amino acid sequence may be compared to areference sequence of at least 18 contiguous nucleotides or 6 amino acidsequences and wherein the portion of the polynucleotide sequence in thecomparison window may comprise additions, deletions, substitutions, andthe like (i.e., gaps) of 20 percent or less as compared to the referencesequence (which does not comprise additions or deletions) for optimalalignment of the two sequences. Optimal alignment of sequences foraligning a comparison window may be conducted by the local homologyalgorithm of Smith and Waterman Adv. Appl. Math. 2:482 (1981), by thehomology alignment algorithm of Needleman and Wunsch J. Mol. Biol.48:443 (1970), by the search for similarity method of Pearson and LipmanProc. Natl. Acad. Sci. (U.S.A.) 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package Release 7.0, (Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), Geneworks, or MacVector softwarepackages), or by inspection, and the best alignment (i.e., resulting inthe highest percentage of homology over the comparison window) generatedby the various methods is selected.

[0126] The term “sequence identity” means that two polynucleotide oramino acid sequences are identical (i.e., on a nucleotide-by-nucleotideor residue-by-residue basis) over the comparison window. The term“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U, or I) or residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the comparison window (i.e., the windowsize), and multiplying the result by 100 to yield the percentage ofsequence identity. The terms “substantial identity” as used hereindenotes a characteristic of a polynucleotide or amino acid sequence,wherein the polynucleotide or amino acid comprises a sequence that hasat least 85 percent sequence identity, preferably at least 90 to 95percent sequence identity, more usually at least 99 percent sequenceidentity as compared to a reference sequence over a comparison window ofat least 18 nucleotide (6 amino acid) positions, frequently over awindow of at least 24-48 nucleotide (8-16 amino acid) positions, whereinthe percentage of sequence identity is calculated by comparing thereference sequence to the sequence which may include deletions oradditions which total 20 percent or less of the reference sequence overthe comparison window. The reference sequence may be a subset of alarger sequence.

[0127] As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis(2^(nd) Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland, Mass. (1991)), which is incorporated herein by reference.Stereoisomers (e.g., D-amino acids) of the twenty conventional aminoacids, unnatural amino acids such as α-, α-disubstituted amino acids,N-alkyl amino acids, lactic acid, and other unconventional amino acidsmay also be suitable components for polypeptides of the presentinvention. Examples of unconventional amino acids include:4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine,ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine,3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and othersimilar amino acids and imino acids (e.g., 4-hydroxyproline). In thepolypeptide notation used herein, the lefthand direction is the aminoterminal direction and the righthand direction is the carboxy-terminaldirection, in accordance with standard usage and convention.

[0128] Similarly, unless specified otherwise, the lefthand end ofsingle-stranded polynucleotide sequences is the 5′ end; the lefthanddirection of double-stranded polynucleotide sequences is referred to asthe 5′ direction. The direction of 5′ to 3′ addition of nascent RNAtranscripts is referred to as the transcription direction; sequenceregions on the DNA strand having the same sequence as the RNA and whichare 5′ to the 5′ end of the RNA transcript are referred to as “upstreamsequences”; sequence regions on the DNA strand having the same sequenceas the RNA and which are 3′ to the 3′ end of the RNA transcript arereferred to as “downstream sequences”.

[0129] As applied to polypeptides, the term “substantial identity” meansthat two peptide sequences, when optimally aligned, such as by theprograms GAP or BESTFIT using default gap weights, share at least 80percent sequence identity, preferably at least 90 percent sequenceidentity, more preferably at least 95 percent sequence identity, andmost preferably at least 99 percent sequence identity. Preferably,residue positions which are not identical differ by conservative aminoacid substitutions. Conservative amino acid substitutions refer to theinterchangeability of residues having similar side chains. For example,a group of amino acids having aliphatic side chains is glycine, alanine,valine, leucine, and isoleucine; a group of amino acids havingaliphatic-hydroxyl side chains is serine and threonine; a group of aminoacids having amide-containing side chains is asparagine and glutamine; agroup of amino acids having aromatic side chains is phenylalanine,tyrosine, and tryptophan; a group of amino acids having basic sidechains is lysine, arginine, and histidine; and a group of amino acidshaving sulfur-containing side chains is cysteine and methionine.Preferred conservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, glutamic-aspartic, and asparagine-glutamine.

[0130] As discussed herein, minor variations in the amino acid sequencesof antibodies or immunoglobulin molecules are contemplated as beingencompassed by the present invention, providing that the variations inthe amino acid sequence maintain at least 75%, more preferably at least80%, 90%, 95%, and most preferably 99%. In particular, conservativeamino acid replacements are contemplated. Conservative replacements arethose that take place within a family of amino acids that are related intheir side chains. Genetically encoded amino acids are generally dividedinto families: (1) acidic=aspartate, glutamate; (2) basic=lysine,arginine, histidine; (3) non-polar=alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan; and (4) unchargedpolar=glycine, asparagine, glutamine, cysteine, serine, threonine,tyrosine. More preferred families are: serine and threonine arealiphatic-hydroxy family; asparagine and glutamine are anamide-containing family; alanine, valine, leucine and isoleucine are analiphatic family; and phenylalanine, tryptophan, and tyrosine are anaromatic family. For example, it is reasonable to expect that anisolated replacement of a leucine with an isoleucine or valine, anaspartate with a glutamate, a threonine with a serine, or a similarreplacement of an amino acid with a structurally related amino acid willnot have a major effect on the binding or properties of the resultingmolecule, especially if the replacement does not involve an amino acidwithin a framework site. Whether an amino acid change results in afunctional peptide can readily be determined by assaying the specificactivity of the polypeptide derivative. Assays are described in detailherein. Fragments or analogs of antibodies or immunoglobulin moleculescan be readily prepared by those of ordinary skill in the art. Preferredamino- and carboxy-termini of fragments or analogs occur near boundariesof functional domains. Structural and functional domains can beidentified by comparison of the nucleotide and/or amino acid sequencedata to public or proprietary sequence databases. Preferably,computerized comparison methods are used to identify sequence motifs orpredicted protein conformation domains that occur in other proteins ofknown structure and/or function. Methods to identify protein sequencesthat fold into a known three-dimensional structure are known. Bowie etal. Science 253:164 (1991). Thus, the foregoing examples demonstratethat those of skill in the art can recognize sequence motifs andstructural conformations that may be used to define structural andfunctional domains in accordance with the invention.

[0131] Preferred amino acid substitutions are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinities, and (4) confer or modify other physicochemical orfunctional properties of such analogs. Analogs can include variousmuteins of a sequence other than the naturally-occurring peptidesequence. For example, single or multiple amino acid substitutions(preferably conservative amino acid substitutions) may be made in thenaturally-occurring sequence (preferably in the portion of thepolypeptide outside the domain(s) forming intermolecular contacts. Aconservative amino acid substitution should not substantially change thestructural characteristics of the parent sequence (e.g., a replacementamino acid should not tend to break a helix that occurs in the parentsequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles (Creighton, Ed., W. H. Freeman andCompany, New York (1984)); Introduction to Protein Structure (C. Brandenand J. T{dot over (o)}oze, eds., Garland Publishing, New York, N.Y.(1991)); and Thornton et at. Nature 354:105 (1991), which are eachincorporated herein by reference.

[0132] The term “polypeptide fragment” as used herein refers to apolypeptide that has an amino-terminal and/or carboxy-terminal deletion,but where the remaining amino acid sequence is identical to thecorresponding positions in the naturally-occurring sequence deduced, forexample, from a full-length cDNA sequence. Fragments typically are atleast 5, 6, 8 or 10 amino acids long, preferably at least 14 amino acidslong, more preferably at least 20 amino acids long, usually at least 50amino acids long, and even more preferably at least 70 amino acids long.The term “analog” as used herein refers to polypeptides which arecomprised of a segment of at least 25 amino acids that has substantialidentity to a portion of a deduced amino acid sequence and which has atleast one of the following properties: (1) specific binding to a EGF-r,under suitable binding conditions, (2) ability to EGF binding to itsreceptor, or (3) ability to inhibit EGF-r expressing cell growth invitro or in vivo. Typically, polypeptide analogs comprise a conservativeamino acid substitution (or addition or deletion) with respect to thenaturally-occurring sequence. Analogs typically are at least 20 aminoacids long, preferably at least 50 amino acids long or longer, and canoften be as long as a full-length naturally-occurring polypeptide.

[0133] Peptide analogs are commonly used in the pharmaceutical industryas non-peptide drus with properties analogous to those of the templatepeptide. These types of non-peptide compound are termed “peptidemimetics” or “peptidomimetics”. Fauchere, J. Adv. Drug Res. 15:29(1986); Veber and Freidinger TINS p.392 (1985); and Evans et al. J Med.Chem. 30:1229 (1987), which are incorporated herein by reference. Suchcompounds are often developed with the aid of computerized molecularmodeling. Peptide mimetics that are structurally similar totherapeutically useful peptides may be used to produce an equivalenttherapeutic or prophylactic effect. Generally, peptidomimetics arestructurally similar to a paradigm polypeptide (i.e., a polypeptide thathas a biochemical property or pharmacological activity), such as humanantibody, but have one or more peptide linkages optionally replaced by alinkage selected from the group consistingof:—CH₂NH—,—CH₂S—,—CH₂—CH₂—,—CH=CH—(cis and trans),—COCH₂—,—CH(OH)CH₂—,and —CH₂SO—, by methods well known in the art. Systematic substitutionof one or more amino acids of a consensus sequence with a D-amino acidof the same type (e.g., D-lysine in place of L-lysine) may be used togenerate more stable peptides. In addition, constrained peptidescomprising a consensus sequence or a substantially identical consensussequence variation may be generated by methods known in the art (Rizoand Gierasch Ann. Rev. Biochem. 61:387 (1992), incorporated herein byreference); for example, by adding internal cysteine residues capable offorming intramolecular disulfide bridges which cyclize the peptide.

[0134] “Antibody” or “antibody peptide(s)” refer to an intact antibody,or a binding fragment thereof that competes with the intact antibody forspecific binding. Binding fragments are produced by recombinant DNAtechniques, or by enzymatic or chemical cleavage of intact antibodies.Binding fragments include Fab, Fab′, F(ab′)₂, Fv, and single-chainantibodies. An antibody other than a “bispecific” or “bifunctional”antibody is understood to have each of its binding sites identical. Anantibody substantially inhibits adhesion of a receptor to acounterreceptor when an excess of antibody reduces the quantity ofreceptor bound to counterreceptor by at least about 20%, 40%, 60% or80%, and more usually greater than about 85% (as measured in an in vitrocompetitive binding assay).

[0135] The term “epitope” includes any protein determinant capable ofspecific binding to an immunoglobulin or T-cell receptor. Epitopicdeterminants usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and usually havespecific three dimensional structural characteristics, as well asspecific charge characteristics. An antibody is said to specificallybind an antigen when the dissociation constant is ≦1 μM, preferably ≦100nM and most preferably ≦10 nM.

[0136] The term “agent” is used herein to denote a chemical compound, amixture of chemical compounds, a biological macromolecule, or an extractmade from biological materials.

[0137] As used herein, the terms “label” or “labeled” refers toincorporation of a detectable marker, e.g., by incorporation of aradiolabeled amino acid or attachment to a polypeptide of biotinylmoieties that can be detected by marked avidin (e.g., streptavidincontaining a fluorescent marker or enzymatic activity that can bedetected by optical or colorimetric methods). In certain situations, thelabel or marker can also be therapeutic. Various methods of labelingpolypeptides and glycoproteins are known in the art and may be used.Examples of labels for polypeptides include, but are not limited to, thefollowing: radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y,⁹⁹Tc, ¹¹¹ In, ¹²⁵I, ¹³¹I), fluorescent labels (e.g., FITC, rhodamine,lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase,β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent,biotinyl groups, predetermined polypeptide epitopes recognized by asecondary reporter (e.g., leucine zipper pair sequences, binding sitesfor secondary antibodies, metal binding domains, epitope tags). In someembodiments, labels are attached by spacer arms of various lengths toreduce potential steric hindrance.

[0138] The term “pharmaceutical agent or drug” as used herein refers toa chemical compound or composition capable of inducing a desiredtherapeutic effect when properly administered to a patient. Otherchemistry terms herein are used according to conventional usage in theart, as exemplified by The McGraw-Hill Dictionary of Chemical Terms(Parker, S., Ed., McGraw-Hill, San Francisco (1985)), incorporatedherein by reference).

[0139] The term “antineoplastic agent” is used herein to refer to agentsthat have the functional property of inhibiting a development orprogression of a neoplasm in a human, particularly a malignant(cancerous) lesion, such as a carcinoma, sarcoma, lymphoma, or leukemia.Inhibition of metastasis is frequently a property of antineoplasticagents.

[0140] As used herein, “substantially pure” means an object species isthe predominant species present (i.e., on a molar basis it is moreabundant than any other individual species in the composition), andpreferably a substantially purified fraction is a composition whereinthe object species comprises at least about 50 percent (on a molarbasis) of all macromolecular species present. Generally, a substantiallypure composition will comprise more than about 80 percent of allmacromolecular species present in the composition, more preferably morethan about 85%, 90%, 95%, and 99%. Most preferably, the object speciesis purified to essential homogeneity (contaminant species cannot bedetected in the composition by conventional detection methods) whereinthe composition consists essentially of a single macromolecular species.

[0141] The term patient includes human and veterinary subjects.

[0142] Antibody Structure

[0143] The basic antibody structural unit is known to comprise atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The amino-terminal portion of eachchain includes a variable region of about 100 to 110 or more amino acidsprimarily responsible for antigen recognition. The carboxy-terminalportion of each chain defines a constant region primarily responsiblefor effector function. Human light chains are classified as kappa andlambda light chains. Heavy chains are classified as mu, delta, gamma,alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgA,and IgE, respectively. Within light and heavy chains, the variable andconstant regions are joined by a “J” region of about 12 or more aminoacids, with the heavy chain also including a “D” region of about 10 moreamino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed.,2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in itsentirety for all purposes). The variable regions of each light/heavychain pair form the antibody binding site.

[0144] Thus, an intact antibody has two binding sites. Except inbifunctional or bispecific antibodies, the two binding sites are thesame.

[0145] The chains all exhibit the same general structure of relativelyconserved framework regions (FR) joined by three hyper variable regions,also called complementarity determining regions or CDRs. The CDRs fromthe two chains of each pair are aligned by the framework regions,enabling binding to a specific epitope. From N-terminal to C-terminal,both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2,FR3, CDR3 and FR4. The assignment of amino acids to each domain is inaccordance with the definitions of Kabat Sequences of Proteins ofImmunological Interest (National Institutes of Health, Bethesda, Md.(1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 196:901-917 (1987);Chothia et al. Nature 342:878-883 (1989).

[0146] A bispecific or bifunctional antibody is an artificial hybridantibody having two different heavy/light chain pairs and two differentbinding sites. Bispecific antibodies can be produced by a variety ofmethods including fusion of hybridomas or linking of Fab′ fragments.See, e.g., Songsivilai & Lachmann Clin. Exp. Immunol. 79: 315-321(1990), Kostelny et al. J. Immunol. 148:1547-1553 (1992). Production ofbispecific antibodies can be a relatively labor intensive processcompared with production of conventional antibodies and yields anddegree of purity are generally lower for bispecific antibodies.Bispecific antibodies do not exist in the form of fragments having asingle binding site (e.g., Fab, Fab′, and Fv).

[0147] Humanization and Display Technologies

[0148] As was discussed above in connection with human antibodygeneration, there are advantages to producing antibodies with reducedimmunogenicity. To a degree, this can be accomplished in connection withtechniques of humanization and display techniques using appropriatelibraries. It will be appreciated that murine antibodies or antibodiesfrom other species can be humanized or primatized using techniques wellknown in the art. See e.g., Winter and Harris Immunol Today 14:43-46(1993) and Wright et al. Crit, Reviews in Immunol. 12125-168 (1992). Theantibody of interest may be engineered by recombinant DNA techniques tosubstitute the CH1, CH2, CH3, hinge domains, and/or the framework domainwith the corresponding human sequence (see WO 92/02190 and U.S. Pat.Nos. 5,530,101, 5,585,089, 5,693,761, 5,693,792, 5,714,350, and5,777,085). Also, the use of Ig cDNA for construction of chimericimmunoglobulin genes is known in the art (Liu et al. P.N.A.S. 84:3439(1987) and J. Immunol. 139:3521 (1987)). mRNA is isolated from ahybridoma or other cell producing the antibody and used to produce cDNA.The cDNA of interest may be amplified by the polymerase chain reactionusing specific primers (U.S. Pat. Nos. 4,683,195 and 4,683,202).Alternatively, a library is made and screened to isolate the sequence ofinterest. The DNA sequence encoding the variable region of the antibodyis then fused to human constant region sequences. The sequences of humanconstant regions genes may be found in Kabat et al. (1991) Sequences ofProteins of Immunological Interest, N.I.H. publication no. 91-3242.Human C region genes are readily available from known clones. The choiceof isotype will be guided by the desired effector functions, such ascomplement fixation, or activity in antibody-dependent cellularcytotoxicity. Preferred isotypes are IgG1, IgG3 and IgG4. Either of thehuman light chain constant regions, kappa or lambda, may be used. Thechimeric, humanized antibody is then expressed by conventional methods.

[0149] Antibody fragments, such as Fv, F(ab′).sub.2 and Fab may beprepared by cleavage of the intact protein, e.g. by protease or chemicalcleavage. Alternatively, a truncated gene is designed. For example, achimeric gene encoding a portion of the F(ab′)₂ fragment would includeDNA sequences encoding the CH1 domain and hinge region of the H chain,followed by a translational stop codon to yield the truncated molecule.

[0150] Consensus sequences of H and L J regions may be used to designoligonucleotides for use as primers to introduce useful restrictionsites into the J region for subsequent linkage of V region segements tohuman C region segments. C region cDNA can be modified by site directedmutagenesis to place a restriction site at the analogous position in thehuman sequence.

[0151] Expression vectors include plasmids, retroviruses, YACs, EBVderived episomes, and the like. A convenient vector is one that encodesa functionally complete human CH or CL immunoglobulin sequence, withappropriate restriction sites engineered so that any VH or VL sequencecan be easily inserted and expressed. In such vectors, splicing usuallyoccurs between the splice donor site in the inserted J region and thesplice acceptor site preceding the human C region, and also at thesplice regions that occur within the human CH exons. Polyadenylation andtranscription termination occur at native chromosomal sites downstreamof the coding regions. The resulting chimeric antibody may be joined toany strong promoter, including retroviral LTRs, e.g. SV-40 earlypromoter, (Okayama et al. Mol. Cell. Bio. 3:280 (1983)), Rous sarcomavirus LTR (Gorman et al. P.N.A.S. 79:6777 (1982)), and moloney murineleukemia virus LTR (Grosschedl et al. Cell 41:885 (1985)); native 1 gpromoters, etc.

[0152] Further, human antibodies or antibodies from other species can begenerated through display-type technologies, including, withoutlimitation, phage display, retroviral display, ribosomal display, andother techniques, using techniques well known in the art and theresulting molecules can be subjected to additional maturation, such asaffinity maturation, as such techniques are well known in the art.Wright and Harris, supra., Hanes and Plucthau PNAS USA 94:4937-4942(1997) (ribosomal display), Parmley and Smith Gene 73:305-318 (1988)(phage display), Scott TIBS 17:241-245 (1992), Cwirla et al. PNAS USA87:6378-6382 (1990), Russel et al. Nucl. Acids Research 21:1081-1085(1993), Hoganboom et al. Immunol. Reviews 130:43-68 (1992), Chiswell andMcCafferty TIBTECH 10:80-84 (1992), and U.S. Pat. No. 5,733,743. Ifdisplay technologies are utilized to produce antibodies that are nothuman, such antibodies can be humanized as described above.

[0153] Using these techniques, antibodies can be generated to EGF-Rexpressing cells, EGF-R itself, forms of EGF-R, epitopes or peptidesthereof, and expression libraries thereto (see e.g. U.S. Pat. No.5,703,057) which can thereafter be screened as described above for theactivities described above.

[0154] Additional Criteria for Antibody Therapeutics

[0155] As discussed herein, the function of the EGF-R antibody appearsimportant to at least a portion of its mode of operation. By function,we mean, by way of example, the activity of the EGF-R antibody inoperation and activity in the costimulatory pathway of EGF-R.Accordingly, in certain respects, it may be desirable in connection withthe generation of antibodies as therapeutic candidates against EGF-Rthat the antibodies be capable of fixing complement and participating inCDC. There are a number of isotypes of antibodies that are capable ofthe same, including, without limitation, the following: murine IgM,murine IgG2a, murine IgG2b, murine IgG3, human IgM, human IgG1, andhuman IgG3. It will be appreciated that antibodies that are generatedneed not initially possess such an isotype but, rather, the antibody asgenerated can possess any isotype and the antibody can be isotypeswitched thereafter using conventional techniques that are well known inthe art. Such techniques include the use of direct recombinanttechniques (see e.g., U.S. Pat. No. 4,816,397), cell-cell fusiontechniques (see e.g., U.S. patent application Ser. No. 08/730,639, filedOct. 11, 1996), among others.

[0156] In the cell-cell fusion technique, a myeloma or other cell lineis prepared that possesses a heavy chain with any desired isotype andanother myeloma or other cell line is prepared that possesses the lightchain. Such cells can, thereafter, be fused and a cell line expressingan intact antibody can be isolated.

[0157] By way of example, the E763 antibody discussed herein is a humananti-EGF-R IgG2 antibody. If such antibody possessed desired binding tothe EGF-R molecule, it could be readily isotype switched to generate ahuman IgM, human IgG 1, or human IgG3 isotype, while still possessingthe same variable region (which defines the antibody's specificity andsome of its affinity). Such molecule would then be capable of fixingcomplement and participating in CDC.

[0158] Accordingly, as antibody candidates are generated that meetdesired “structural” attributes as discussed above, they can generallybe provided with at least certain of the desired “functional” attributesthrough isotype switching.

[0159] Design and Generation of Other Therapeutics

[0160] In accordance with the present invention and based on theactivity of the antibodies that are produced and characterized hereinwith respect to EGF-R, the design of other therapeutic modalities beyondantibody moieties is facilitated. Such modalities include, withoutlimitation, advanced antibody therapeutics, such as bispecificantibodies, immunotoxins, and radiolabeled therapeutics, generation ofpeptide therapeutics, gene therapies, particularly intrabodies,antisense therapeutics, and small molecules.

[0161] In connection with the generation of advanced antibodytherapeutics, where complement fixation is a desirable attribute, it maybe possible to sidestep the dependence on complement for cell killingthrough the use of bispecifics, immunotoxins, or radiolabels, forexample.

[0162] For example, in connection with bispecific antibodies, bispecificantibodies can be generated that comprise (i) two antibodies one with aspecificity to EGF-R and another to a second molecule that areconjugated together, (ii) a single antibody that has one chain specificto EGF-R and a second chain specific to a second molecule, or (iii) asingle chain antibody that has specificity to EGF-R and the othermolecule. Such bispecific antibodies can be generated using techniquesthat are well known for example, in connection with (i) and (ii) seee.g., Fanger et al. Immunol Methods 4:72-81 (1994) and Wright andHarris, supra. and in connection with (iii) see e.g., Traunecker et al.Int. J. Cancer (Suppl.) 7:51-52 (1992). In each case, the secondspecificity can be made to the heavy chain activation receptors,including, without limitation, CD16 or CD64 (see e.g., Deo et al. 18:127(1997)) or CD89 (see e.g., Valerius et al. Blood 90:4485-4492 (1997)).Bispecific antibodies prepared in accordance with the foregoing would belikely to kill cells expressing EGF-R, and particularly those cells inwhich the EGF-R antibodies of the invention are effective.

[0163] In connection with immunotoxins, antibodies can be modified toact as immunotoxins utilizing techniques that are well known in the art.See e.g., Vitetta Immunol Today 14:252 (1993). See also U.S. Pat. No.5,194,594. In connection with the preparation of radiolabeledantibodies, such modified antibodies can also be readily preparedutilizing techniques that are well known in the art. See e.g., Junghanset al. in Cancer Chemotherapy and Biotherapy 655-686 (2d edition,Chafner and Longo, eds., Lippincott Raven (1996)). See also U.S. Pat.Nos. 4,681,581, 4,735,210, 5,101,827, 5,102,990 (RE 35,500), 5,648,471,and 5,697,902. Each of immunotoxins and radiolabeled molecules would belikely to kill cells expressing EGF-R, and particularly those cells inwhich the antibodies of the invention are effective.

[0164] In connection with the generation of therapeutic peptides,through the utilization of structural information related to EGF-R andantibodies thereto, such as the antibodies of the invention (asdiscussed below in connection with small molecules) or screening ofpeptide libraries, therapeutic peptides can be generated that aredirected against EGF-R. Design and screening of peptide therapeutics isdiscussed in connection with Houghten et al. Biotechniques 13:412-421(1992), Houghten PNAS USA 82:5131-5135 (1985), Pinalla et al.Biotechniques 13:901-905 (1992), Blake and Litzi-Davis BioConjugateChem. 3:510-513 (1992). Immunotoxins and radiolabeled molecules can alsobe prepared, and in a similar manner, in connection with peptidicmoieties as discussed above in connection with antibodies.

[0165] Assuming that the EGF-R molecule (or a form, such as a splicevariant or alternate form) is functionally active in a disease process,it will also be possible to design gene and antisense therapeuticsthereto through conventional techniques. Such modalities can be utilizedfor modulating the function of EGF-R. In connection therewith theantibodies of the present invention facilitate design and use offunctional assays related thereto. A design and strategy for antisensetherapeutics is discussed in detail in International Patent ApplicationNo. WO 94/29444. Design and strategies for gene therapy are well known.However, in particular, the use of gene therapeutic techniques involvingintrabodies could prove to be particularly advantageous. See e.g., Chenet al. Human Gene Therapy 5:595-601 (1994) and Marasco Gene Therapy4:11-15 (1997). General design of and considerations related to genetherapeutics is also discussed in International Patent Application No.WO 97/38137.

[0166] Small molecule therapeutics can also be envisioned in accordancewith the present invention. Drugs can be designed to modulate theactivity of EGF-R based upon the present invention. Knowledge gleanedfrom the structure of the EGF-R molecule and its interactions with othermolecules in accordance with the present invention, such as theantibodies of the invention, and others can be utilized to rationallydesign additional therapeutic modalities. In this regard, rational drugdesign techniques such as X-ray crystallography, computer-aided (orassisted) molecular modeling (CAMM), quantitative or qualitativestructure-activity relationship (QSAR), and similar technologies can beutilized to focus drug discovery efforts. Rational design allowsprediction of protein or synthetic structures which can interact withthe molecule or specific forms thereof which can be used to modify ormodulate the activity of EGF-R. Such structures can be synthesizedchemically or expressed in biological systems. This approach has beenreviewed in Capsey et al. Genetically Engineered Human Therapeutic Drugs(Stockton Press, NY (1988)). Further, combinatorial libraries can bedesigned and sythesized and used in screening programs, such as highthroughput screening efforts.

[0167] Therapeutic Administration and Formulations

[0168] It will be appreciated that administration of therapeuticentities in accordance with the invention will be administered withsuitable carriers, excipients, and other agents that are incorporatedinto formulations to provide improved transfer, delivery, tolerance, andthe like. A multitude of appropriate formulations can be found in theformulary known to all pharmaceutical chemists: Remington'sPharmaceutical Sciences (15^(th) ed, Mack Publishing Company, Easton,Pa. (1975)), particularly Chapter 87 by Blaug, Seymour, therein. Theseformulations include, for example, powders, pastes, ointments, jellies,waxes, oils, lipids, lipid (cationic or anionic) containing vesicles(such as Lipofectin™), DNA conjugates, anhydrous absorption pastes,oil-in-water and water-in-oil emulsions, emulsions carbowax(polyethylene glycols of various molecular weights), semi-solid gels,and semi-solid mixtures containing carbowax. Any of the foregoingmixtures may be appropriate in treatments and therapies in accordancewith the present invention, provided that the active ingredient in theformulation is not inactivated by the formulation and the formulation isphysiologically compatible and tolerable with the route ofadministration.

[0169] Preparation of Antibodies

[0170] Antibodies in accordance with the invention are preferablyprepared through the utilization of a transgenic mouse that has asubstantial portion of the human antibody producing genome inserted butthat is rendered deficient in the production of endogenous, murine,antibodies. Such mice, then, are capable of producing humanimmunoglobulin molecules and antibodies and are deficient in theproduction of murine immunoglobulin molecules and antibodies.Technologies utilized for achieving the same are disclosed in thepatents, applications, and references disclosed in the Background,herein. In particular, however, a preferred embodiment of transgenicproduction of mice and antibodies therefrom is disclosed in U.S. patentapplication Ser. No. 08/759,620, filed Dec. 3, 1996, the disclosure ofwhich is hereby incorporated by reference. See also Mendez et al. NatureGenetics 15:146-156 (1997), the disclosure of which is herebyincorporated by reference.

[0171] Through use of such technology, we have produced fully humanmonoclonal antibodies to a variety of antigens. Essentially, we immunizeXenoMouse™ lines of mice with an antigen of interest, recover lymphaticcells (such as B-cells) from the mice that express antibodies, fuse suchrecovered cells with a myeloid-type cell line to prepare immortalhybridoma cell lines, and such hybridoma cell lines are screened andselected to identify hybridoma cell lines that produce antibodiesspecific to the antigen of interest. We utilized these techniques inaccordance with the present invention for the preparation of antibodiesspecific to EGF-r. Herein, we describe the production of eight hybridomacell lines that produce antibodies specific to EGF-r. Further, weprovide a characterization of the antibodies produced by such celllines, including nucleotide and amino acid sequence analyses of theheavy and light chains of such antibodies.

[0172] The hybridoma cell lines discussed herein are designated E1.1,E2.4, E2.5, E6.2, E6.4, E2.1 1, E6.3, and E7.6.3. Each of the antibodiesproduced by the aforementioned cell lines are fully human IgG2 heavychains with human kappa light chains. In general, antibodies inaccordance with the invention possess very high affinities, typicallypossessing Kd's of from about 10⁻⁹ through about 10⁻¹¹ M, when measuredby either solid phase and solution phase.

[0173] As will be appreciated, antibodies in accordance with the presentinvention can be expressed in cell lines other than hybridoma celllines. Sequences encoding particular antibodies can be used fortransformation of a suitable mammalian host cell. Transformation can beby any known method for introducing polynucleotides into a host cell,including, for example packaging the polynucleotide in a virus (or intoa viral vector) and transducing a host cell with the virus (or vector)or by transfection procedures known in the art, as exemplified by U.S.Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455 (which patentsare hereby incorporated herein by reference). The transformationprocedure used depends upon the host to be transformed. Methods forintroduction of heterologous polynucleotides into mammalian cells arewell known in the art and include dextran-mediated transfection, calciumphosphate precipitation, polybrene mediated transfection, protoplastfusion, electroporation, encapsulation of the polynucleotide(s) inliposomes, and direct microinjection of the DNA into nuclei.

[0174] Mammalian cell lines available as hosts for expression are wellknown in the art and include many immortalized cell lines available fromthe American Type Culture Collection (ATCC), including but not limitedto Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney(BHK) cells, monkey kidney cells (COS), human hepatocellular carcinomacells (e.g., Hep G2), and a number of other cell lines. Cell lines ofparticular preference are selected through determining which cell lineshave high expression levels and produce antibodies with constitutiveEGF-r binding properties.

[0175] Antibodies in accordance with the present invention are potentinhibitors of EGF and TGF-a binding to its receptor, EGF-r. Such resultsare discussed in Examples 5 and 6 and shown in FIGS. 35 through 38.Consistent with such results, and as shown in FIG. 39 and discussed inconnection with Example 7, antibodies in accordance with the presentinvention also inhibit the growth of certain human carcinoma cell linesin vitro. Antibodies in accordance with the present invention alsoprevent the growth of certain human carcinomas in vivo. Such results areshown in FIGS. 40 through 42 and discussed in connection with Example 8.In Example 9, we demonstrate that antibodies in accordance with thepresent invention, at least in combination with an antineoplastic agent,will eradicate an existing tumor in an animal. Moreover, antibodytherapy, as a monotherapy (i.e., not in combination with anantineoplastic agent) appears possible in accordance with the antibodiesin accordance with the present invention, where it did not appearpossible in the prior art, for example through the use of the antibody225. Such results are discussed in connection with Example 9 and shownin FIGS. 43-44.

[0176] The results demonstrated in accordance with the present inventionindicate that antibodies in accordance with the present inventionpossess certain qualities that may make the present antibodies moreefficacious than current therapeutic antibodies against EGF-r, e.g.,225. The 225 antibody in clinical development by Imclone is a chimericIgG1 antibody with an affinity of 2×10⁻¹⁰ M, which, while appearingefficacious in combination therapy with an antineoplastic agent, doesnot appear very efficacious in monotherapy. In contrast, antibodies inaccordance with the invention (and particularly the E2.5 and E7.6.3antibodies of the invention) have significantly higher affinities(E2.5:1.6×10⁻¹¹ M; E7.6.3:5.7×10⁻¹¹ M) and appear efficacious inmonotherapy in addition to combination therapy with an antineoplasticagent and at lower doses than with the C225 antibody.

EXAMPLES

[0177] The following examples, including the experiments conducted andresults achieved are provided for illustrative purposes only and are notto be construed as limiting upon the present invention.

EXAMPLE 1 Generation of Anti-EGF-r-Antibody Producing Hybridomas

[0178] Antibodies of the invention were prepared, selected, and assayedin accordance with the present Example.

[0179] Immunization and Hybridoma Generation:

[0180] XenoMice (8 to 10 weeks old) were immunized intraperitoneallywith 2×10⁷ A431 (ATCC CRL-7907) cells resuspended in phosphate bufferedsaline (PBS). This dose was repeated three times. Four days beforefusion, the mice received a final injection of cells in PBS. Spleen andlymph node lymphocytes from immunized mice were fused with thenon-secretory myeloma NSO-bc12 line (Ray and Diamond, 1994) and weresubjected to HAT selection as previously described (Galfre and Milstein,1981). A large panel of hybridomas all secreting EGF-r specific humanIgG₂K (as detected below) antibodies were recovered. As described inExample 2, certain of the antibodies selected from the panel wereselected for their ability to compete with the 225 antibody.EGFr-specific hybridomas were identified by ELISA using purified A431cell membrane-derived EGFr protein (Sigma, St. Louis, MO, E3641). Largequantities of antibodies were purified from ascites, derived from SCIDmice inoculated with antibody-producing hybridomas, using protein-Aaffinity chromatography.

[0181] ELISA Assay:

[0182] ELISA for determination of antigen-specific antibodies in mouseserum and in hybridoma supernatants was carried out as described(Coligan et al., 1994) using affinity-purified EGF-r from A431 cells(Sigma, E-3641) to capture the antibodies. The concentrations of humanand mouse immunoglobulins were determined using the following captureantibodies: rabbit anti-human IgG (Southern Biotechnology, 6145-01),goat anti-human Igκ (Vector Laboratories, AI-3060), mouse anti-human IgM(CGI/ATCC, HB-57), for human gamma, kappa, and mu Ig, respectively, andgoat anti-mouse IgG (Caltag, M 30100), goat anti-mouse Igκ(SouthernBiotechnology, 1050-01), goat anti-mouse IgM (Southern Biotechnology,1020-01), and goat anti-mouse λ(Southern Biotechnology, 1060-01) tocapture mouse gamma, kappa, mu, and lambda Ig, respectively. Thedetection antibodies used in ELISA experiments were goat anti-mouseIgG-HRP (Caltag, M-30107), goat anti-mouse Igκ-HRP (Caltag, M 33007),mouse anti-human IgG2-HRP (Southern Biotechnology, 9070-05), mouseanti-human IgM-HRP (Southern Biotechnology, 9020-05), and goatanti-human kappa-biotin (Vector, BA-3060). Standards used forquantitation of human and mouse Ig were: human IgG₂κ (Calbiochem,400122), human IgMκ (Cappel, 13000), mouse IgGκ (Cappel 55939), mouseIgMκ (Sigma, M-3795), and mouse IgG₃λ (Sigma, M-9019).

[0183] Determination of Affinity Constants of Fully Human Mabs byBIAcore:

[0184] Affinity measurement of purified human monoclonal antibodies, Fabfragments, or hybridoma supernatants by plasmon resonance was carriedout using the BIAcore 2000 instrument, using general procedures outlinedby the manufacturers.

[0185] Based upon the general procedures outlined by the manufacture,kinetic analyses of the antibodies were performed using antigensimmobilized onto the sensor surface at a low density. Soluble EGF-rpurified from A431 cell membranes (Sigma, E-3641) or the recombinantextracellular domain of EGFr (20) immobilized onto the sensor surfacewas generally used at a surface density of between about 228 and 303 RU.The dissociation (kd or k_(off)) and association (ka or k_(on)) rateswere determined using the software provided by the manufacturer (BIAevaluation 2.1). Affinity measurements of antibody in solution werecarried out as described (18).

[0186] Determination of Affinity Constants in Solution by ELISA:

[0187] In order to determine antibody binding affinity in solution byELISA, various concentrations of the monoclonal antibodies to EGF-r wereincubated with EGF-r at a constant concentration until equilibrium wasreached. Thereafter, the concentration of the free EGF-r in the reactionsolution was determined by an indirect ELISA. Accordingly, themonoclonal antibodies at concentrations of between 3.0×10⁻¹¹ M through2.7×10⁻⁷ M were incubated with EGF-r at a concentration of 4×10⁻¹⁰ M in200 μl of PBS with 0.5% BSA for 15 hrs at room temperature. Afterincubation, 70 μl of each mixture was transferred into the wells of96-well microtiter plates previously coated with the same monoclonalantibody (100 μl/well, at 2 μg/ml in coating buffer) and incubated for15 min at room temperature. After washing with washing buffer, the EGF-rretained on the plate was detected by mouse anti-EGF-r-HRP, which bindsto the carbohydrate of the EGF-r protein. The concentration of EGF-r wascalculated against its standard and used for the calculation of boundand free antibodies in the original antigen-antibody reaction solution.The binding affinity of each monoclonal antibody to EGF-r was calculatedusing Scatchard analysis.

[0188] Receptor Binding Assays:

[0189] The EGF receptor binding assay was carried out with 6 A431 cellsor SW948 cells (0.4×10 cells per well) which were incubated with varyingconcentrations of antibodies in PBS binding buffer for 30 minutes at 4°C. 0.1 nM [¹²⁵I]EGF (Amersham, IM-196) or [¹²⁵I]TGF-α (Amersham) wasadded to each well, and the plates were incubated for 90 min at 4° C.The plates were washed five times, air-dried and counted in ascintillation counter. Anti-EGF-r mouse antibodies 225 and 528(Calbiochem) were used as controls.

[0190] EGFr binding assays were also conducted using human recombinant[¹²⁵I]EGF or [¹²⁵I]TGFα (Amersham Life Science, Arlington Heights, Ill.)as previously described (Mendez). Briefly, human carcinoma cells growingin Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calfserum (FCS) were detached with trypsin, washed with phosphate-bufferedsaline (PBS) and resuspended in binding buffer (PBS containing 0.1%bovine serum albumin (Sigma) and 0.02% NaN₃), and distributed in 96-wellMultiscreen filter plates (Millipore) at 4.0×10⁵ cells/well in 50 μl.Fully human anti-EGFr or control anti-KLH MAbs, control human myelomaIgG₂κMab (Calbiochem, Cambridge, Mass., 400122), or mouse anti-EGFr 225or 528 MAbs (Calbiochem, GR13 or GR14), diluted in binding buffer, wereadded in 50 μl aliquots per well. Plates were incubated for 30 min at 4°C. [¹²⁵I]-EGF or [¹²⁵]TGFA (0.1 μCi/well in 50 μl) was added and theplates were further incubated for 90 min at 4° C. After incubation, theplates were washed five times with binding buffer, air-dried and countedin a scintillation counter. The percentage of specifically bound[¹²⁵I]EGF or [¹²⁵I]TGFα was calculated as the mean cpm detected in thepresence of antibody divided by cpm detected in the presence of bufferonly. The binding data obtained was fitted using GraphPad Prism(GraphPad Software, Inc. San Diego, Calif.).

EXAMPLE 2 Co-Selection of Anti-EGF-r-Antibodies with the m225 Antibody

[0191] As discussed above, the antibody 225 has been demonstrated topossess a high affinity for, and effective inhibition of the binding ofEGF and TGF-α to EGF-r. Thus, we expected that if we selected humanantibodies against EGF-r that are prepared in accordance with thepresent invention with the antibody 225 in a competition assay,antibodies to the same or similar epitope to which the 225 antibodybinds would be selected.

[0192] Accordingly, we conducted BIAcore assays in which soluble EGF-rpurified from A431 cell membranes (Sigma, E-3641) was pretreated withthe antibody 225 and thereafter treated with antibodies of theinvention. Where antibodies of the invention did not bind, suchantibodies of the invention were screened for binding affinity asdescribed above.

[0193] In the following Table, affinity measurements for certain of theantibodies selected in this manner are provided: TABLE I Solid Phase In(by BIAcore) Solution Surface By ELISA Hybri- k_(on) K_(off) K_(D)Density KD doma (M⁻¹S⁻¹) (S⁻¹) (M) [RU] (M) E1.1 2.3 × 10⁶ 1.7 × 10⁻⁴7.6 × 10⁻¹¹ 228 1.1 × 10⁻¹⁰ E2.4 2.8 × 10⁶ 9.78 × 10⁻⁵  3.5 × 10⁻¹¹ 8181.1 × 10⁻¹⁰ E2.5 1.2 × 10⁶ 1.9 × 10⁻⁵ 1.6 × 10⁻¹¹ 228 3.6 × 10⁻¹⁰ E2.111.9 × 10⁶ 3.0 × 10⁻⁴ 1.6 × 10⁻¹⁰ 228 1.1 × 10⁻¹⁰ E7.6.3 2.0 × 10⁶ 1.1 ×10⁻⁴ 5.7 × 10⁻¹¹ 228 ND

[0194] As will be observed, antibodies selected in this manner possessexceptionally high affinities and binding constants.

EXAMPLE 3 Structures of Anti-EGF-r-Antibodies Prepared in Accordancewith the Invention

[0195] In the following discussion, structural information related toantibodies prepared in accordance with the invention is provided.

[0196] In order to analyze structures of antibodies produced inaccordance with the invention, we cloned genes encoding the heavy andlight chain fragments out of the particular hybridoma. Gene cloning andsequencing was accomplished as follows:

[0197] Poly(A)⁺mRNA was isolated from approximately 2×10⁵ hybridomacells derived from immunized XenoMice using a Fast-Track kit(Invitrogen). The generation of random primed cDNA was followed by PCR.Human V_(H) or human V_(κ) family specific variable region primers(Marks et. al., 1991) or a universal human VH primer, MG-30(CAGGTGCAGCTGGAGCAGTCIGG) was used in conjunction with primers specificfor the human Cγ2 constant region (MG-40d;5′-GCTGAGGGAGTAGAGTCCTGAGGA-3′) or Cκ constant region (hκP2; aspreviously described in Green et al., 1994). Sequences of humanMabs-derived heavy and kappa chain transcripts from hybridomas wereobtained by direct sequencing of PCR products generated from poly(A+)RNA using the primers described above. PCR products were also clonedinto pCRII using a TA cloning kit (Invitrogen) and both strands weresequenced using Prism dye-terminator sequencing kits and an ABI 377sequencing machine. All sequences were analyzed by alignments to the “VBASE sequence directory” (Tomlinson et al., MRC Centre for ProteinEngineering, Cambridge, UK) using MacVector and Geneworks softwareprograms.

[0198] Hybridoma E1.1

[0199] The antibody secreted by the hybridoma E1.1 comprises a humanIgG2 antibody having a human kappa light chain. The antibodies wereanalyzed for structural information related to their heavy chain andlight chain gene utilization, as well as their amino acid sequences.Thus, heavy chain VH, D, and JH and light chain VK and JK geneutilization was analyzed and differences between the coded product andthe particular gene utilization was also analyzed. Accordingly, theantibody secreted by the hybridoma E1.1 evidenced the following geneutilization:

[0200] V_(H)—4-31

[0201] D—2

[0202] J_(H)—5

[0203] Vκ—018

[0204] Jκ—4

[0205] As reported in the V BASE sequence directory, the amino acidsequence encoded by the V_(H) 4-31 gene was determined to be:

[0206]VSGGSISSGGYYWSWIRQHPGKGLEWIGYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR(SEQ ID NO:1)

[0207] As reported in the V BASE sequence directory, the amino acidsequence encoded by the Vκ (018) gene was determined to be:

[0208]TITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLP(SEQ ID NO:2)

[0209] Amino acid and nucleotide sequence information respecting theheavy and light chains are provided below in connection with FIGS. 1-4.FIG. 1 is an amino acid sequence of a heavy chain immunoglobulinmolecule that is secreted by the hybridoma E1.1. Differences between thesequence encoded by heavy chain variable gene 4-31 and the sequence ofthe E1.1 secreted heavy chain are indicated in bold and enlarged font.The contiguous sequence from CDR1 through CDR3 is indicated byunderlining and CDR1, CDR2, and CDR3 sequences are each indicated bydouble underlining.

[0210]FIG. 2 is a nucleotide sequence of the cDNA encoding the heavychain immunoglobulin molecule of FIG. 1 that was cloned out of thehybridoma El. 1.

[0211]FIG. 3 is an amino acid sequence of a kappa light chainimmunoglobulin molecule that is secreted by the hybridoma E1.1.Differences between the sequence encoded by light chain variable gene018 and the sequence of the E1.1 secreted light chain are indicated inbold and enlarged font. The contiguous sequence from CDR1 through CDR3is indicated by underlining and CDR1, CDR2, and CDR3 sequences are eachindicated by double underlining.

[0212]FIG. 4 is a nucleotide sequence of the cDNA encoding the kappalight chain immunoglobulin molecule of FIG. 3 that was cloned out of thehybridoma E1.1.

[0213] Hybridoma E2.4

[0214] The antibody secreted by the hybridoma E2.4 comprises a humanIgG2 antibody having a human kappa light chain. The antibodies wereanalyzed for structural information related to their heavy chain andlight chain gene utilization, as well as their amino acid sequences.Thus, heavy chain V_(H), D, and J_(H) and light chain Vκ and Jκ geneutilization was analyzed and differences between the coded product andthe particular gene utilization was also analyzed. Accordingly, theantibody secreted by the hybridoma E2.4 evidenced the following geneutilization:

[0215] V_(H)—4-31

[0216] D—A1/A4

[0217] J_(H)—3

[0218] Vκ—018

[0219] Jκ—4

[0220] Amino acid and nucleotide sequence information respecting theheavy and light chains are provided below in connection with FIGS. 5-8.FIG. 5 is an amino acid sequence of a heavy chain immunoglobulinmolecule that is secreted by the hybridoma E2.4. Differences between thesequence encoded by heavy chain variable gene 4-31 and the sequence ofthe E2.4 secreted heavy chain are indicated in bold and enlarged font.The contiguous sequence from CDR1 through CDR3 is indicated byunderlining and CDR1, CDR2, and CDR3 sequences are each indicated bydouble underlining.

[0221]FIG. 6 is a nucleotide sequence of the cDNA encoding the heavychain immunoglobulin molecule of FIG. 5 that was cloned out of thehybridoma E2.4.

[0222]FIG. 7 is an amino acid sequence of a kappa light chainimmunoglobulin molecule that is secreted by the hybridoma E2.4.Differences between the sequence encoded by light chain variable gene018 and the sequence of the E2.4 secreted light chain are indicated inbold and enlarged font. The contiguous sequence from CDR1 through CDR3is indicated by underlining and CDR1, CDR2, and CDR3 sequences are eachindicated by double underlining.

[0223]FIG. 8 is a nucleotide sequence of the cDNA encoding the kappalight chain immunoglobulin molecule of FIG. 7 that was cloned out of thehybridoma E2.4.

[0224] Hybridoma E2.5

[0225] The antibody secreted by the hybridoma E2.5 comprises a humanIgG2 antibody having a human kappa light chain. The antibodies wereanalyzed for structural information related to their heavy chain andlight chain gene utilization, as well as their amino acid sequences.Thus, heavy chain V_(H), D, and J_(H) and light chain Vκ and Jκ geneutilization was analyzed and differences between the coded product andthe particular gene utilization was also analyzed. Accordingly, theantibody secreted by the hybridoma E2.5 evidenced the following geneutilization:

[0226] V_(H)—4-31

[0227] D—XP1/21-10

[0228] J_(H)—4

[0229] Vκ—018

[0230] Jκ—2

[0231] Amino acid and nucleotide sequence information respecting theheavy and light chains are provided below in connection with FIGS. 9-12.FIG. 9 is an amino acid sequence of a heavy chain immunoglobulinmolecule that is secreted by the hybridoma E2.5. Differences between thesequence encoded by heavy chain variable gene 4-31 and the sequence ofthe E2.5 secreted heavy chain are indicated in bold and enlarged font.The contiguous sequence from CDR1 through CDR3 is indicated byunderlining and CDR1, CDR2, and CDR3 sequences are each indicated bydouble underlining.

[0232]FIG. 10 is a nucleotide sequence of the cDNA encoding the heavychain immunoglobulin molecule of FIG. 9 that was cloned out of thehybridoma E2.5.

[0233]FIG. 11 is an amino acid sequence of a kappa light chainimmunoglobulin molecule that is secreted by the hybridoma E2.5.Differences between the sequence encoded by light chain variable gene018 and the sequence of the E2.5 secreted light chain are indicated inbold and enlarged font. The contiguous sequence from CDR1 through CDR3is indicated by underlining and CDR1, CDR2, and CDR3 sequences are eachindicated by double underlining.

[0234]FIG. 12 is a nucleotide sequence of the cDNA encoding the kappalight chain immunoglobulin molecule of FIG. 11 that was cloned out ofthe hybridoma E2.5.

[0235] Hybridoma E6.2

[0236] The antibody secreted by the hybridoma E6.2 comprises a humanIgG2 antibody having a human kappa light chain. The antibodies wereanalyzed for structural information related to their heavy chain andlight chain gene utilization, as well as their amino acid sequences.Thus, heavy chain V_(H), D, and J_(H) and light chain Vκ and Jκ geneutilization was analyzed and differences between the coded product andthe particular gene utilization was also analyzed. Accordingly, theantibody secreted by the hybridoma E6.2 evidenced the following geneutilization:

[0237] V_(H)—4-31

[0238] D—?(CNTCCCTT)

[0239] J_(H)—6

[0240] Vκ—018

[0241] Jκ—1

[0242] Amino acid and nucleotide sequence information respecting theheavy and light chains are provided below in connection with FIGS.13-16. FIG. 13 is an amino acid sequence of a heavy chain immunoglobulinmolecule that is secreted by the hybridoma E6.2. Differences between thesequence encoded by heavy chain variable gene 4-31 and the sequence ofthe E6.2 secreted heavy chain are indicated in bold and enlarged font.The contiguous sequence from CDR1 through CDR3 is indicated byunderlining and CDR1, CDR2, and CDR3 sequences are each indicated bydouble underlining.

[0243]FIG. 14 is a nucleotide sequence of the cDNA encoding the heavychain immunoglobulin molecule of FIG. 13 that was cloned out of thehybridoma E6.2.

[0244]FIG. 15 is an amino acid sequence of a kappa light chainimmunoglobulin molecule that is secreted by the hybridoma E6.2.Differences between the sequence encoded by light chain variable gene018 and the sequence of the E6.2 secreted light chain are indicated inbold and enlarged font. The contiguous sequence from CDR1 through CDR3is indicated by underlining and CDR1, CDR2, and CDR3 sequences are eachindicated by double underlining.

[0245]FIG. 16 is a nucleotide sequence of the cDNA encoding the kappalight chain immunoglobulin molecule of FIG. 15 that was cloned out ofthe hybridoma E6.2.

[0246] Hybridoma E6.4

[0247] The antibody secreted by the hybridoma E6.4 comprises a humanIgG2 antibody having a human kappa light chain. The antibodies wereanalyzed for structural information related to their heavy chain andlight chain gene utilization, as well as their amino acid sequences.Thus, heavy chain V_(H), D, and J_(H) and light chain Vκ, and Jκ geneutilization was analyzed and differences between the coded product andthe particular gene utilization was also analyzed. Accordingly, theantibody secreted by the hybridoma E6.4 evidenced the following geneutilization:

[0248] V_(H)—4-31

[0249] D—A1/A4

[0250] J_(H)—4

[0251] Vκ—012

[0252] Jκ—2

[0253] As reported in the V BASE sequence directory, the amino acidsequence encoded by the Vκ 012 gene was determined to be:

[0254]TITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTP(SEQ ID NO:36)

[0255] Amino acid and nucleotide sequence information respecting theheavy and light chains are provided below in connection with FIGS.17-20. FIG. 17 is an amino acid sequence of a heavy chain immunoglobulinmolecule that is secreted by the hybridoma E6.4. Differences between thesequence encoded by heavy chain variable gene 4-31 and the sequence ofthe E6.4 secreted heavy chain are indicated in bold and enlarged font.The contiguous sequence from CDR1 through CDR3 is indicated byunderlining and CDRI, CDR2, and CDR3 sequences are each indicated bydouble underlining.

[0256]FIG. 18 is a nucleotide sequence of the cDNA encoding the heavychain immunoglobulin molecule of FIG. 17 that was cloned out of thehybridoma E6.4.

[0257]FIG. 19 is an amino acid sequence of a kappa light chainimmunoglobulin molecule that is secreted by the hybridoma E6.4.Differences between the sequence encoded by light chain variable gene012 and the sequence of the E6.4 secreted light chain are indicated inbold and enlarged font. The contiguous sequence from CDR1 through CDR3is indicated by underlining and CDR1, CDR2, and CDR3 sequences are eachindicated by double underlining.

[0258]FIG. 20 is a nucleotide sequence of the cDNA encoding the kappalight chain immunoglobulin molecule of FIG. 19 that was cloned out ofthe hybridoma E6.4.

[0259] Hybridoma E2.11

[0260] The antibody secreted by the hybridoma E2.11 comprises a humanIgG2 antibody having a human kappa light chain. The antibodies wereanalyzed for structural information related to their heavy chain andlight chain gene utilization, as well as their amino acid sequences.Thus, heavy chain VH, D, and JH and light chain VK and JK geneutilization was analyzed and differences between the coded product andthe particular gene utilization was also analyzed. Accordingly, theantibody secreted by the hybridoma E2.11 evidenced the following geneutilization:

[0261] V_(H)—4-61

[0262] D—XP1/21-10

[0263] J_(H)—4

[0264] Vκ—018

[0265] Jκ—4

[0266] As reported in the V BASE sequence directory, the amino acidsequence encoded by the V_(H) 4-61 gene was determined to be:

[0267] VSGGSVSSGSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR (SEQ ID NO:23)

[0268] Amino acid and nucleotide sequence information respecting theheavy and light chains are provided below in connection with FIGS.21-24. FIG. 21 is an amino acid sequence of a heavy chain immunoglobulinmolecule that is secreted by the hybridoma E2.11. Differences betweenthe sequence encoded by heavy chain variable gene 4-61 and the sequenceof the E2.11 secreted heavy chain are indicated in bold and enlargedfont. The contiguous sequence from CDR1 through CDR3 is indicated byunderlining and CDR1, CDR2, and CDR3 sequences are each indicated bydouble underlining.

[0269]FIG. 22 is a nucleotide sequence of the cDNA encoding the heavychain immunoglobulin molecule of FIG. 21 that was cloned out of thehybridoma E2.11.

[0270]FIG. 23 is an amino acid sequence of a kappa light chainimmunoglobulin molecule that is secreted by the hybridoma E2.11.Differences between the sequence encoded by light chain variable gene018 and the sequence of the E2.11 secreted light chain are indicated inbold and enlarged font. The contiguous sequence from CDR1 through CDR3is indicated by underlining and CDR1, CDR2, and CDR3 sequences are eachindicated by double underlining.

[0271]FIG. 24 is a nucleotide sequence of the cDNA encoding the kappalight chain immunoglobulin molecule of FIG. 23 that was cloned out ofthe hybridoma E2.11.

[0272] Hybridoma E6.3

[0273] The antibody secreted by the hybridoma E6.3 comprises a humanIgG2 antibody having a human kappa light chain. The antibodies wereanalyzed for structural information related to their heavy chain andlight chain gene utilization, as well as their amino acid sequences.Thus, heavy chain V_(H), D, and J_(H) and light chain Vκ and Jκ geneutilization was analyzed and differences between the coded product andthe particular gene utilization was also analyzed. Accordingly, theantibody secreted by the hybridoma E6.3 evidenced the following geneutilization:

[0274] V_(H)—4-61

[0275] D—1-2rc

[0276] J_(H)—4

[0277] Vκ—018

[0278] Jκ−4

[0279] Amino acid and nucleotide sequence information respecting theheavy and light chains are provided below in connection with FIGS.25-28. FIG. 25 is an amino acid sequence of a heavy chain immunoglobulinmolecule that is secreted by the hybridoma E6.3. Differences between thesequence encoded by heavy chain variable gene 4-61 and the sequence ofthe E6.3 secreted heavy chain are indicated in bold and enlarged font.The contiguous sequence from CDR1 through CDR3 is indicated byunderlining and CDR1, CDR2, and CDR3 sequences are each indicated bydouble underlining.

[0280]FIG. 26 is a nucleotide sequence of the cDNA encoding the heavychain immunoglobulin molecule of FIG. 25 that was cloned out of thehybridoma E6.3.

[0281]FIG. 27 is an amino acid sequence of a kappa light chainimmunoglobulin molecule that is secreted by the hybridoma E6.3.Differences between the sequence encoded by light chain variable gene018 and the sequence of the E6.3 secreted light chain are indicated inbold and enlarged font. The contiguous sequence from CDR1 through CDR3is indicated by underlining and CDR1, CDR2, and CDR3 sequences are eachindicated by double underlining.

[0282]FIG. 28 is a nucleotide sequence of the cDNA encoding the kappalight chain immunoglobulin molecule of FIG. 27 that was cloned out ofthe hybridoma E6.3.

[0283] Hybridoma E7.6.3

[0284] The antibody secreted by the hybridoma E7.6.3 comprises a humanIgG2 antibody having a human kappa light chain. The antibodies wereanalyzed for structural information related to their heavy chain andlight chain gene utilization, as well as their amino acid sequences.Thus, heavy chain V_(H), D, and J_(H) and light chain Vκ and Jκ geneutilization was analyzed and differences between the coded product andthe particular gene utilization was also analyzed. Accordingly, theantibody secreted by the hybridoma E7.6.3 evidenced the following geneutilization:

[0285] V_(H)−4-61

[0286] D—XP4rc-XP1

[0287] J_(H)—3

[0288] Vκ—018

[0289] Jκ—4

[0290] Amino acid and nucleotide sequence information respecting theheavy and light chains are provided below in connection with FIGS.29-32. FIG. 29 is an amino acid sequence of a heavy chain immunoglobulinmolecule that is secreted by the hybridoma E7.6.3. Differences betweenthe sequence encoded by heavy chain variable gene 4-61 and the sequenceof the E7.6.3 secreted heavy chain are indicated in bold and enlargedfont. The contiguous sequence from CDR1 through CDR3 is indicated byunderlining and CDR1, CDR2, and CDR3 sequences are each indicated bydouble underlining.

[0291]FIG. 30 is a nucleotide sequence of the cDNA encoding the heavychain immunoglobulin molecule of FIG. 29 that was cloned out of thehybridoma E7.6.3.

[0292]FIG. 31 is an amino acid sequence of a kappa light chainimmunoglobulin molecule that is secreted by the hybridoma E7.6.3.Differences between the sequence encoded by light chain variable gene018 and the sequence of the E7.6.3 secreted light chain are indicated inbold and enlarged font. The contiguous sequence from CDR1 through CDR3is indicated by underlining and CDR1, CDR2, and CDR3 sequences are eachindicated by double underlining.

[0293]FIG. 32 is a nucleotide sequence of the cDNA encoding the kappalight chain immunoglobulin molecule of FIG. 31 that was cloned out ofthe hybridoma E7.6.3.

[0294] The following antibodies that are secreted by hybridomas E20.1,E20.3, E20.8.1, E20.11.2, E20.18, E20.19.2, E20.21, E20.22, E7.5.2, andE7.8.2 bind to EGFr, but do not compete with E7.6.3 for binding to EGFr.

[0295] Hybridoma E20.1

[0296] The antibody secreted by the hybridoma E20.1 comprises a humanIgG2 antibody having a human kappa light chain. The antibodies wereanalyzed for structural information related to their heavy chain andlight chain gene utilization, as well as their amino acid sequences.Thus, heavy chain V_(H), D, and J_(H) and light chain Vκ and Jκ geneutilization was analyzed and differences between the coded product andthe particular gene utilization was also analyzed. Accordingly, theantibody secreted by the hybridoma E20.1 evidenced the following geneutilization:

[0297] V_(H)—DP-50 (3-33)

[0298] D—DXP4

[0299] J_(H)—JH4b

[0300] Vκ—LFVK431

[0301] JKκ—JK3

[0302] The amino acid sequences encoded by the V_(H) DP-50 (3-33) geneand Vκ LFVK431 gene are available in the V BASE sequence directory.

[0303] Amino acid and nucleotide sequence information respecting theheavy and light chains are provided below in connection with FIGS.57-58. FIG. 57 shows a nucleotide sequence of the cDNA encoding theheavy chain immunoglobulin molecule that was cloned out of the hybridomaE20.1 and the corresponding amino acid sequence of a heavy chainimmunoglobulin molecule that is secreted by the hybridoma E20.1.

[0304]FIG. 58 shows a nucleotide sequence of the cDNA encoding the lightchain immunoglobulin molecule that was cloned out of the hybridoma E20.1and the corresponding amino acid sequence of a light chainimmunoglobulin molecule that is secreted by the hybridoma E20.1.

[0305] Hybridoma E20.3

[0306] The antibody secreted by the hybridoma E20.3 comprises a humanIgG2 antibody having a human kappa light chain. The antibodies wereanalyzed for structural information related to their heavy chain andlight chain gene utilization, as well as their amino acid sequences.Thus, heavy chain V_(H), D, and J_(H) and light chain Vκ and Jκ geneutilization was analyzed and differences between the coded product andthe particular gene utilization was also analyzed. Accordingly, theantibody secreted by the hybridoma E20.3 evidenced the following geneutilization:

[0307] V_(H)—DP15 (1-8)

[0308] D—DN1

[0309] J_(H)—JH4b

[0310] Vκ—B3/DPK24

[0311] Jκ—JK4

[0312] The amino acid sequences encoded by the V_(H) DP-15 (1-8) geneand Vκ Vκ B3/DPK24 gene are available in the V BASE sequence directory.

[0313] Amino acid and nucleotide sequence information respecting theheavy and light chains are provided below in connection with FIGS.59-60. FIG. 59 shows a nucleotide sequence of the cDNA encoding theheavy chain immunoglobulin molecule that was cloned out of the hybridomaE20.3 and the corresponding amino acid sequence of a heavy chainimmunoglobulin molecule that is secreted by the hybridoma E20.3.

[0314]FIG. 60 shows a nucleotide sequence of the cDNA encoding the lightchain immunoglobulin molecule that was cloned out of the hybridoma E20.3and the corresponding amino acid sequence of a light chainimmunoglobulin molecule that is secreted by the hybridoma E20.3.

[0315] Hybridoma E208.1

[0316] The antibody secreted by the hybridoma E20.8.1 comprises a humanIgG2 antibody having a human kappa light chain. The antibodies wereanalyzed for structural information related to their heavy chain andlight chain gene utilization, as well as their amino acid sequences.Thus, heavy chain V_(H), D, and J_(H) and light chain Vκ and Jκ geneutilization was analyzed and differences between the coded product andthe particular gene utilization was also analyzed. Accordingly, theantibody secreted by the hybridoma E20.8.1 evidenced the following geneutilization:

[0317] V_(H)—DP-50 (3-33)

[0318] D—D1/D21-9/D23-7

[0319] J_(H)—JH4b

[0320] Vκ—B3/DPK24

[0321] Jκ—JK2

[0322] Amino acid and nucleotide sequence information respecting theheavy and light chains are provided below in connection with FIGS.61-62. FIG. 61 shows a nucleotide sequence of the cDNA encoding theheavy chain immunoglobulin molecule that was cloned out of the hybridomaE20.8.1 and the corresponding amino acid sequence of a heavy chainimmunoglobulin molecule that is secreted by the hybridoma E20.8.1.

[0323]FIG. 62 shows a nucleotide sequence of the cDNA encoding the lightchain immunoglobulin molecule that was cloned out of the hybridomaE20.8.1 and the corresponding amino acid sequence of a light chainimmunoglobulin molecule that is secreted by the hybridoma E20.8.1.

[0324] Hybridoma E20.11.2

[0325] The antibody secreted by the hybridoma 20.11.2 comprises a humanIgG2 antibody having a human kappa light chain. The antibodies wereanalyzed for structural information related to their heavy chain andlight chain gene utilization, as well as their amino acid sequences.Thus, heavy chain V_(H), D, and J_(H) and light chain Vκ and Jκ geneutilization was analyzed and differences between the coded product andthe particular gene utilization was also analyzed. Accordingly, theantibody secreted by the hybridoma E20.11.2 evidenced the following geneutilization:

[0326] V_(H)—DP-50 (3-33)

[0327] D—DIR5

[0328] J_(H)—JH4b

[0329] Vκ—B3/DPK24

[0330] Jκ—JK1

[0331] Amino acid and nucleotide sequence information respecting theheavy and light chains are provided below in connection with FIGS.63-64. FIG. 63 shows a nucleotide sequence of the cDNA encoding theheavy chain immunoglobulin molecule that was cloned out of the hybridomaE20.11.2 and the corresponding amino acid sequence of a heavy chainimmunoglobulin molecule that is secreted by the hybridoma E20.11.2.

[0332]FIG. 64 shows a nucleotide sequence of the cDNA encoding the lightchain immunoglobulin molecule that was cloned out of the hybridomaE20.11.2 and the corresponding amino acid sequence of a light chainimmunoglobulin molecule that is secreted by the hybridoma E20.11.2.

[0333] Hybridoma E20.18

[0334] The antibody secreted by the hybridoma E20.18 comprises a humanIgG2 antibody having a human kappa light chain. The antibodies wereanalyzed for structural information related to their heavy chain andlight chain gene utilization, as well as their amino acid sequences.Thus, heavy chain V_(H), D, and J_(H) and light chain Vκ and Jκ geneutilization was analyzed and differences between the coded product andthe particular gene utilization was also analyzed. Accordingly, theantibody secreted by the hybridoma E20. 18 evidenced the following geneutilization:

[0335] V_(H)—DP-50 (3-33)

[0336] D—**

[0337] J_(H)—**

[0338] Vκ—B3/DPK24

[0339] Jκ—JK2

[0340] Amino acid and nucleotide sequence information respecting theheavy and light chains are provided below in connection with FIGS.65-66. FIG. 65 shows a nucleotide sequence of the cDNA encoding theheavy chain immunoglobulin molecule that was cloned out of the hybridomaE20. 18 and the corresponding amino acid sequence of a heavy chainimmunoglobulin molecule that is secreted by the hybridoma E20.18.

[0341]FIG. 66 shows a nucleotide sequence of the cDNA encoding the lightchain immunoglobulin molecule that was cloned out of the hybridomaE20.18 and the corresponding amino acid sequence of a light chainimmunoglobulin molecule that is secreted by the hybridoma E20.18.

[0342] Hybridoma E20.19.2

[0343] The antibody secreted by the hybridoma E20.19.2 comprises a humanIgG2 antibody having a human kappa light chain. The antibodies wereanalyzed for structural information related to their heavy chain andlight chain gene utilization, as well as their amino acid sequences.Thus, heavy chain V_(H), D, and J_(H) and light chain Vκ and Jκ geneutilization was analyzed and differences between the coded product andthe particular gene utilization was also analyzed. Accordingly, theantibody secreted by the hybridoma E20.19.2 evidenced the following geneutilization:

[0344] V_(H)—DP-71 (4-59)

[0345] D—**

[0346] J_(H)—JH4b

[0347] Vκ—B3/DPK24

[0348] Jκ—JK1

[0349] The amino acid sequence encoded by the V_(H) DP-71 (4-59) gene isavailable in the V BASE sequence directory.

[0350] Amino acid and nucleotide sequence information respecting theheavy and light chains are provided below in connection with FIGS.67-68. FIG. 67 shows a nucleotide sequence of the cDNA encoding theheavy chain immunoglobulin molecule that was cloned out of the hybridomaE20.19.2 and the corresponding amino acid sequence of a heavy chainimmunoglobulin molecule that is secreted by the hybridoma E20.19.2.

[0351]FIG. 68 shows a nucleotide sequence of the cDNA encoding the lightchain immunoglobulin molecule that was cloned out of the hybridomaE20.19.2 and the corresponding amino acid sequence of a light chainimmunoglobulin molecule that is secreted by the hybridoma E20.19.2.

[0352] Hybridoma E20.21

[0353] The antibody secreted by the hybridoma E20.21 comprises a humanIgG2 antibody having a human kappa light chain. The antibodies wereanalyzed for structural information related to their heavy chain andlight chain gene utilization, as well as their amino acid sequences.Thus, heavy chain V_(H), D, and J_(H) and light chain Vκ and Jκ geneutilization was analyzed and differences between the coded product andthe particular gene utilization was also analyzed. Accordingly, theantibody secreted by the hybridoma E20.21 evidenced the following geneutilization:

[0354] V_(H)—DP-65 (4-31)

[0355] D—DIR3

[0356] J_(H)—JH6b

[0357] Vκ—LFVK431

[0358] Jκ—JK3

[0359] Amino acid and nucleotide sequence information respecting theheavy chain is provided below in connection with FIG. 69. FIG. 69 showsa nucleotide sequence of the cDNA encoding the heavy chainimmunoglobulin molecule that was cloned out of the hybridoma E20.21 andthe corresponding amino acid sequence of a heavy chain immunoglobulinmolecule that is secreted by the hybridoma E20.21.

[0360] Hybridoma E20.22

[0361] The antibody secreted by the hybridoma E20.22 comprises a humanIgG2 antibody having a human kappa light chain. The antibodies wereanalyzed for structural information related to their heavy chain andlight chain gene utilization, as well as their amino acid sequences.Thus, heavy chain V_(H), D, and J_(H) and light chain Vκ and Jκ geneutilization was analyzed and differences between the coded product andthe particular gene utilization was also analyzed. Accordingly, theantibody secreted by the hybridoma E20.22 evidenced the following geneutilization:

[0362] V_(H)—DP-71 (4-59)

[0363] D—DIR4

[0364] J_(H)—JH6b

[0365] Vκ—??

[0366] Jκ—??

[0367] Amino acid and nucleotide sequence information respecting theheavy chain is provided below in connection with FIG. 70. FIG. 70 showsa nucleotide sequence of the cDNA encoding the heavy chainimmunoglobulin molecule that was cloned out of the hybridoma E20.22 andthe corresponding amino acid sequence of a heavy chain immunoglobulinmolecule that is secreted by the hybridoma E20.22.

[0368] Hybridoma E7.5.2

[0369] The antibody secreted by the hybridoma E7.5.2 comprises a humanIgG2 antibody having a human kappa light chain. The antibodies wereanalyzed for structural information related to their heavy chain andlight chain gene utilization, as well as their amino acid sequences.Thus, heavy chain VH, D, and JH and light chain VK and JK geneutilization was analyzed and differences between the coded product andthe particular gene utilization was also analyzed. Accordingly, theantibody secreted by the hybridoma E7.5.2 evidenced the following geneutilization:

[0370] V_(H)—DP-75 (1-2)

[0371] D—A1/A1rc

[0372] J_(H)—JH4

[0373] Vκ—02

[0374] Jκ_(—JK)2

[0375] The sequence of the VH1-2 (DP-75) VK 02 gene products areavailable in the V BASE sequence directory. The nucleotide and aminoacid sequences of the heavy and light chains of the E7.5.2 antibody areprovided in FIGS. 72 and 73.

[0376] Hybridoma E7.8.2

[0377] The antibody secreted by the hybridoma E7.8.2 comprises a humanIgG2 antibody having a human kappa light chain. The antibodies wereanalyzed for structural information related to their heavy chain andlight chain gene utilization, as well as their amino acid sequences.Thus, heavy chain V_(H), D, and J_(H) and light chain Vκ and Jκ geneutilization was analyzed and differences between the coded product andthe particular gene utilization was also analyzed. Accordingly, theantibody secreted by the hybridoma E7.8.2 evidenced the following geneutilization:

[0378] V_(H)—DP-75 (1-2)

[0379] D—1/IR-K1

[0380] J_(H)—JH4

[0381] Vκ012

[0382] Jκ—JK2

EXAMPLE 4 Analysis of Heavy and Light Chain Amino Acid Substitutions

[0383]FIG. 33 provides a comparison of specific anti-EGF-r antibodyheavy chain amino acid sequence comparisons with the amino acid sequenceof the particular V_(H) gene which encodes the heavy chain of theparticular antibody. FIG. 34 provides a similar comparison of specificanti-EGF-r antibody light chain amino acid sequence comparisons with theamino acid sequence of the particular VK gene which encodes the lightchain of the particular antibody. As will be observed, there are severalremarkably conserved amino acid substitutions amongst the heavy andlight chain sequences. In particular, in the heavy chains of theantibodies, all of the heavy chain molecules are encoded by V_(H) 4family genes and have a Glycine in position 10 in V_(H) 4-31 encodedantibodies and Serine in position 10 in V_(H) 4-61 encoded antibodiesare each substituted with an Aspartic Acid. Also in the V_(H) 4-31 heavychains, all but one of the antibodies includes a Serine in position 7substitution to Asparagine. A similar, though not quite as predominantsubstitution is observed in position 35, where a Serine in two of theV_(H) 4-31 encoded antibodies and two of the V_(H) 4-61 encodedantibodies is substituted with an Asparagine. Also, in two of the V_(H)4-31 encoded antibodies and two of the V_(H) 4-61 encoded antibodiesthere are substitutions at position 28, where in each case, a Tyrosineis substituted with a Serine (E2.4) or a Histidine (E6.4, E2.11, andE7.6.3). Five of the antibodies, three of the V_(H) 4-31 encodedantibodies and two of the V_(H) 4-61 encoded antibodies, possess Valineto Leucine (E2.4 and E2.11) or Isoleucine (E2.5, E6.2, and E7.6.3) atposition 50.

[0384] In connection with the kappa light chains amino acid sequences,all of the sequences are encoded by Vκ I family genes, with seven of themolecules being encoded by 018 genes and one (E6.4) being encoded by an012 gene. There is a high degree of homology between the 012 and 018gene products, as evidenced when the E6.4 molecule is compared with the018 gene product, along with the other molecules, in FIG. 34. The E6.4molecule possesses only two substitutions relative to the 012 geneproduct, as shown in FIG. 19, and only 13 substitutions relative to the018 gene product. All of the antibodies possess a substitution atposition 74 in CDR3 where an Asparagine is substituted with a Serine(E1.1, E2.5, E2.11, and E6.3), Histidine (E2.4, E6.2, and E7.6.3), orArginine (E6.4). The remainder of the substitutions are less highlyconserved. However, a number of the antibodies appear to possesssubstitutions within the CDR'S. However, it is interesting to note thatE7.6.3, which is an antibody with very high affinities, possesses noamino acid substitutions in the light chain amino acid sequence untiljust proximal to CDR3 and within CDR3.

[0385] It will be appreciated that each of the above-identified aminoacid substitutions exist in close proximity to or within a CDR. Suchsubstitutions would appear to bear some effect upon the binding of theantibody to the EGF receptor molecule. Further, such substitutions couldhave significant effect upon the affinity of the antibodies.

[0386] As was discussed above, anti-EGF-r antibodies have beendemonstrated to possess certain anti-tumor activities. The followingexperiments were carried out in order to determine if antibodies inaccordance with the present invention possessed such anti-tumoractivities.

EXAMPLE 5 Blockage of EGF and TGF-α Binding to Human EpidermoidCarcinoma A431 Cells by Human Anti-EGF-r Antibodies in vitro

[0387] An in vitro assay was conducted to determine if antibodies inaccordance with the present invention were capable of blocking EGFbinding to a human carcinoma cell line. The experiment was conducted tocompare the binding of antibodies in accordance with the invention withthe murine monoclonal antibody 225 which, as discussed above, haspreviously demonstrated anti-cancer activity.

[0388] In this example, the human epidermoid carcinoma A431 cell linewas utilized. The A431 cell line is known for its high expression levelof EGF-r (about 2×10⁶ EGF-r molecules per cell). Therefore, higherconcentrations of anti-EGF-r antibodies are required to saturate all ofthe binding sites. The results from this example are shown in FIG. 35.In the Figure, blockage of I¹²⁵ labeled EGF binding to human epidermoidcarcinoma A431 cells by a human anti-EGF-r antibody in vitro isdemonstrated. In the Figure, (□) depicts the results achieved by theanti-EGF-r antibody in accordance with the invention (E7.6.3), (∘)depicts the results achieved by the murine monoclonal antibody 225, and(▴) depicts the results achieved by a control, nonspecific, human IgG2antibody.

[0389]FIG. 36 shows inhibition of EGF binding to human epidermoidcarcinoma A431 cells by a panel of human anti-EGF-r antibodies inaccordance with the invention in vitro when compared to the 225, 528,and nonspecific human IgG2 controls. In the Figure, (□) depicts theresults achieved by the murine monoclonal antibody 225, (∘) depicts theresults achieved by the murine monoclonal antibody 528, (▾) depicts theresults achieved using the E1.1 antibody in accordance with theinvention, (▴) depicts the results achieved using the E2.4 antibody inaccordance with the invention, (

)depicts the results achieved using the E2.5 antibody in accordance withthe invention, (z,901 ) depicts the results achieved using the E2.6antibody in accordance with the invention, (♦) depicts the resultsachieved using the E2.11 antibody in accordance with the invention, and

depicts the results achieved using a control, nonspecific human IgG2antibody.

[0390] The results indicate that antibodies in accordance with theinvention may block EGF binding to surface expressed EGF-r on A431 cellsbetter than the 225 and 528 antibodies. Antibodies in accordance withthe invention appear to begin inhibiting binding at an 8 nMconcentration as compared to a 10 nM concentration for the 225 antibody.

[0391] In connection with inhibition of TGF-α binding, similar efficacyis observed through use of antibodies in accordance with the inventionwhen compared to the 225 antibody. FIG. 37 shows inhibition of TGF-αbinding to human epidermoid carcinoma A431 cells by human anti-EGF-rantibodies in vitro, where (□) depicts the results achieved by themurine monoclonal antibody 225, (♦) depicts the results achieved usingthe E6.2 antibody in accordance with the invention, () depicts theresults achieved using the E6.3 antibody in accordance with theinvention, (▴) depicts the results achieved using the E7.2 antibody inaccordance with the invention, (▪) depicts the results achieved usingthe E7. 10 antibody in accordance with the invention, (▾) depicts theresults achieved using the E7.6.3, and ({circle over (X)}) depicts theresults achieved using a control, nonspecific human IgG2 antibody.

[0392] The results indicate that antibodies in accordance with theinvention may block TGF-α binding to surface expressed EGF-r on A431cells better than the 225 antibody. Antibodies in accordance with theinvention appear to begin inhibiting binding at an 0.1 nM concentrationas compared to a 1 nM concentration for the 225 antibody.

EXAMPLE 6 Blockage of EGF Binding to Human Colon Adenocarcinoma SW948Cells by Human Anti-EGF-r Antibodies in vitro

[0393] Another in vitro assay was conducted to determine if antibodiesin accordance with the present invention were capable of blocking EGFbinding to yet another human carcinoma cell line. The experiment wasconducted to compare the binding of antibodies in accordance with theinvention with the murine monoclonal antibody 225 which, as discussedabove, has previously demonstrated anti-cancer activity.

[0394] In this example, the human colon adenocarcinoma SW948 cell linewas utilized. In contrast to the A431 cell line, the SW948 cell line hasrelatively low expression of EGF-r on its surface (about 40,000molecules per cell). Therefore, less of the anti-EGF-r antibodies arerequired to saturate all of the binding sites of the receptors on thecells. The results from this example are shown in FIG. 38. In theFigure, blockage of I¹²⁵ labeled EGF binding to human colonadenocarcinoma SW948 cells by a human anti-EGF-r antibody in vitro isdemonstrated. In the Figure, (◯) depicts the results achieved by ananti-EGF-r antibody in accordance with the invention (E7.6.3), (□)depicts the results achieved by the murine monoclonal antibody 225, and(▴) depicts the results achieved by a control, nonspecific, human IgG2antibody.

[0395] The results indicate that the antibody in accordance with theinvention blocks EGF binding to SW948 cells at least as well as the 225antibody. In fact, the curve is slightly improved with respect to theantibody in accordance with the invention, showing inhibition at lowerconcentrations than the 225 antibody.

EXAMPLE 7 Inhibition of Human Colon Adenocarcinoma SW948 Cell Growth byHuman Anti-EGF-r Antibodies in vitro

[0396] We also conducted an in vitro assay to determine whether and towhat degree, as compared to the 225 antibody, antibodies in accordancewith the invention were capable of inhibiting cancer cell growth. Theexperiment was conducted to compare the inhibition by antibodies inaccordance with the invention with the inhibition by the murinemonoclonal antibody 225 which, as discussed above, has previouslydemonstrated anti-cancer activity.

[0397] In this example, the human colon adenocarcinoma SW948 cell linewas utilized. In our hands, only the SW948 cell line showedEGF-dependent cell growth. In contrast, the A431 cell line showed growthinhibition in the presence of EGF in vitro. The results are shown inFIG. 39 where it is demonstrated that human anti-EGF-r antibodies inaccordance with the present invention inhibit the growth of SW948 cellsin vitro. In the Figure, (◯) depicts the results achieved by ananti-EGF-r antibody in accordance with the invention (E7.6.3), (□)depicts the results achieved by the murine monoclonal antibody 225, and(▴) depicts the results achieved by a control, nonspecific, human IgG2antibody.

[0398] The results indicate that the antibody in accordance with theinvention inhibits growth of SW948 cells at least as well as the 225antibody. In fact, the curve is slightly improved with respect to theantibody in accordance with the invention, showing an apparent 100%inhibition in cell growth at approximately 100 μg/ml whereas the 225antibody appears to plateau at an inhibition level between 80 to 90% inthe same dosage range.

EXAMPLE 8 Inhibition of Human Epidermoid Carcinoma Growth in Nude Miceby Human Anti-EGF-r Antibodies in vivo

[0399] In the present experiment, we sought to determine if antibodiesin accordance with the present invention were capable of inhibitingtumor cell growth in vivo. In the experiment, nude mice at the age of 8weeks were inoculated subcutaneously with the human epidermoid carcinomaA431 cell line. Mice were injected with 5×10⁶ A431 cells. One of twodosages of an antibody in accordance with the invention or one of twocontrols was injected intraperitoneally on the same day when the A431cells were inoculated. Three adminstrations of either antibody orcontrol followed and mice were followed for subcutaneous tumor formationand size. The dosages of antibody utilized were either 1.0 mg or 0.2 mg.The controls were either phosphate buffered saline or a nonspecifichuman IgG2 antibody.

[0400] The results from this experiment are shown in FIG. 40. In theFigure, the inhibition of human epidermoid carcinoma cell growth in nudemice through use of human anti-EGF-r antibodies in accordance with theinvention in vivo is evident. In the Figure, (▴) depicts the resultsachieved with a dosage of 1.0 mg of a human anti-EGF-r antibody inaccordance with the present invention (E7.6.3) (n=5), (▾)depicts theresults achieved with a dosage of 0.2 mg of the E.7.6.3 antibody (n=4),(□) depicts the results achieved by a control, nonspecific, human IgG2antibody (n=6), and (◯) depicts the results achieved utilizing phosphatebuffered saline as a control (n=6).

[0401] No tumor growth was observed in animals treated with the E7.6.3antibody whereas control animals grew significant tumors within 25 daysof tumor cell inoculation.

[0402] In the same experiment, three antibodies in accordance with theinvention were compared. The results are shown in FIG. 41. Each of theantibodies in accordance with the present invention, E7.6.3 at 1 mg in 5mice and 0.2 mg in 4 mice, E2.5 at 1 mg in 3 mice and 0.2 mg in 3 mice,and E1.1 at 1 mg in 3 mice, demonstrated inhibition of the humanepidermoid carcinoma formation in the mice in comparison to controls.All of the control animals (including 6 PBS-treated animals and 6 humanIgG2-treated animals) developed significant tumors within 19 days ofinoculation whereas none of the the animals treated with the humananti-EGF-r antibodies in accordance with the invention developed tumorswithin 19 days of inoculation.

[0403]FIG. 42 shows the results of following the animals from thisabove-mentioned same experiment for 130 days post inoculation with thehuman epidermoid carcinoma. The results from this experiment are shownin FIG. 42. In the Figure, it will be observed that all of the controlmice had developed tumors within 20 days of tumor cell inoculation. Incontrast, the first mouse treated with an antibody in accordance withthe present invention to develop a tumor was on day 70. By day 130, only4 out of 15 of the experimental animals had developed tumors.Interestingly, none of the experimental animals treated with the 0.2 mgdosage of the E2.5 antibody developed tumors within the test period.

[0404] The above experiment in connection with this Example 8demonstrate that antibodies in accordance with the present invention ifadministered contemporaneously with the inoculation of a tumor cell lineappear to almost entirely prevent the initiation of tumor cell growthand initiation of the tumor. Moreover, it will be observed that theinhibitory effect on tumor cell growth appears long-lasting.

EXAMPLE 9 Eradication of Human Epidermoid Carcinoma Growth in Nude Miceby Human Anti-EGF-r Antibodies in vivo

[0405] While preventing tumor cell growth and/or establishment of atumor, as discussed above in connection with the preceding example, is apositive finding, from a therapeutic point of view, eradication of anestablished tumor is also highly desirable. Accordingly, in thefollowing experiments we examined whether antibodies in accordance withthe invention were capable of eradicating an established tumor in amammal. Previous data generated in connection with the 225 antibodyindicated that in order to effectively eradicate an established tumorthrough use of the 225 antibody it was necessary to complement treatmentwith an antineoplastic agent. Thus, in connection with our experiments,we looked at antibody treatment both alone and in combination withantineoplastic agent treatment.

[0406] In the experiment, nude mice were inoculated subcutaneously with5×10⁶ A431 human epidermoid carcinoma cells on day 0. Mice were treatedwith either antibodies, chemotherapeutic agents, and/or controls afterthe tumor had an opportunity to become established (size≧0.4 cm³).Treatments were begun and continued on days 5, 8, 10, 14, 16, and 21,with chemotherapies being administered only on days 5 and 6. Therapiesconsisted of an antibody in accordance with the invention (E7.6.3), theantineoplastic agent doxorubicin, and a combination of antibody anddoxorubicin. Controls were phosphate buffered saline or a nonspecifichuman IgG2 antibody. Each treatment group consisted of 5 animals. Thedata generated from the experiments are shown in FIG. 43, where (▴)depicts the results achieved with a dosage of 1 mg of a human anti-EGF-rantibody in accordance with the present invention (E7.6.3) (n=5), (×)depicts the results achieved with a dosage of 125 μg of doxorubicin, (*)depicts the results achieved with a dosage of 1 mg of a human anti-EGF-rantibody in accordance with the present invention (E7.6.3) incombination with a dosage of 125 μg of doxorubicin, (▪) depicts theresults achieved by a control, nonspecific, human IgG2 antibody, and (♦)depicts the results achieved utilizing phosphate buffered saline as acontrol.

[0407] As will be observed, administration of the E7.6.3 antibody incombination with doxorubicin resulted in complete eradication tumorgrowth. Further, tumor growth was completely arrested throughadministration of the E7.6.3 antibody alone.

[0408] In a similar experiment, the results of which are shown in FIG.44, following inoculation with the tumor, five mice were treated with0.5 mg of the E2.5 antibody on days 5, 8, 10, 14, 16, and 21 and fivemice were treated with a combination of the E2.5 antibody administeredon days 5, 8, 10, 14, 16, and 21 and doxorubicin administered on days 5and 6. In the Figure, (♦) depicts the results achieved with a dosage of0.5 mg of a human anti-EGF-r antibody in accordance with the presentinvention (E2.5), (▪) depicts the results achieved with a dosage of 125μg of doxorubicin, (▴) depicts the results achieved with a dosage of 0.5mg of a human anti-EGF-r antibody in accordance with the presentinvention (E2.5) in combination with a dosage of 125 μg of doxorubicin,(×) depicts the results achieved utilizing phosphate buffered saline asa control, and (*) depicts the results achieved utilizing a control,nonspecific, human IgG2 antibody.

[0409] As will be observed, administration of the E2.5 antibody byitself, or in combination with doxorubicin, resulted in near completeeradication of tumors in the mice.

EXAMPLE 10 Additional Characterization of Antibodies in Accordance withthe Invention

[0410] In order to further characterize antibodies in accordance withthe invention, we conducted a number of additional in vitro and in vivoassays. In addition to the assays discussed above, certain of suchassays were conducted in accordance with the following Materials andMethods:

[0411] A. Materials and Methods

[0412] In Vitro Tumor Cell Proliferation Assay:

[0413] The effect of antibodies on the growth of human tumor cells wasdetermined using the method described by Ishiyama et al. (21). Briefly,2×10³ cells in 100 μl of DMEM:F12 growth medium without serum wereseeded into each well of a 96-well plate. Aliquots of each dilutedantibody (100 μl/well) were added in triplicate to the wells and thecultures were incubated at 37° C. for 5 days. The controls consisted ofeither medium alone or medium containing dilutions of an human myelomaIgG₂κ control antibodies. After incubation, the medium was removed fromeach well by aspiration. All cells were fixed with 0.25% glutaraldehyde,then washed in 0.9% NaCl, air-dried and stained with Crystal Violet(Fisher Scientific, Pittsburgh, Pa.) for 15 min a t room temperature.After washing with tap water and air-drying, 100 μl of methanol wasadded to each well and the A₅₉₅ of each supernatant was determined in aSpectra Max spectrophotometer (Molecular Devices, Sunnyvale, Calif.).The percentage of growth inhibition is calculated as the me an A₅₉₅measured in medium only minus A₅₉₅ in the presence of antibody dividedby mean A₅₉₅ in the presence of medium only.

[0414] Measurement of Cell Activation By Cytosensor Microphysiometry:

[0415] To assess the effect of antibody on EGF-mediated signaling,Cytosensor microphysiometry (Molecular Devices, Sunnyvale Calif.) wasused. The Cytosensor detects early biochemical events in cell activationbased upon increases in the rate of acid release by the cells (22). Acidrelease was measured as described in the user's manual provided byMolecular Devices, Inc. Briefly, A431 cells (5×10⁴) were seeded in 1 mlmedium in a Cytosensor cell capsule and cultured at 37° C. for 24 h.After incubation, the cell capsules were assembled and loaded in theCytosensor sensing chamber maintained at 37° C. The chamber was perfused(50 μl/min) with low buffer RPMI 1640 medium containing 1 mM sodiumphosphate and 1 mg/ml endotoxin-free bovine serum albumin. Acid releaserates were determined with 30-s potentiometric pH measurements after a85-s pump cycle and 5-s delay (120-s total cycle time). Basal acidrelease rates ranged from 60 to 120 mV per second. % inhibition iscalculated as the acid release in the presence of EGF only minus theacid release in the presence of EGF and antibody divided by the acidrelease in the presence of EGF only.

[0416] Tumor Xenograft Mouse Models:

[0417] Male BALB/c-nu/nu mice (6-8 weeks of age) were injected s.c. with5×10⁶ A431 or MDA-MB-468 (ATCC, HTB-132) cells in 100 μl PBS. Tumorssizes were measured twice a week with a vernier caliper and tumor volumewas calculated as the product of length×width×height×π/6. Mice withestablished tumors were randomly divided into treatment groups. Animalswere treated with antibodies using different regimens. Typically, micereceived antibody treatment twice a week over three consecutive weekseither concomitant with the tumor cell injection (prophylactictreatment) or after tumor establishment (therapeutic treatment). Themice were followed for tumor xenograft growth and survival for at least60 days.

[0418] Tumor Histopathological Evaluation:

[0419] Biopsies obtained from athymic mice carrying human xenograftswere fixed in 10% neutral buffered formalin, embedded in paraffin, andsectioned. The sections were then stained with hematoxylin and eosin, asdescribed (23).

[0420] EGFr Phosphorylation:

[0421] 70% confluent A431 cells were pre-incubated at a lowconcentration of fetal bovine serum (0.5%) overnight in 37° C. The cellswere then treated with 16 nM EGF in the presence or absence of differentconcentrations of E7.6.3 MAb for 30 minutes at 37° C. After the 30-minincubation, the cells were washed three times with cold PBS and scrapedinto 0.5 ml of lysis buffer (10 mM Tris, 150 mM NaCl, 5 mM EDTA, 1%Triton-100, 0.1 mg/ml PMSF, 1 μg/ml aprotinin, 1 g/ml leupeptin and 1 mMsodium orthovanadate). After 30 minutes of incubation on ice, the lysatewas centrifuged at 10,000 rpm for 5 minutes in an Eppendorfmicrocentrifuge at 4° C. The protein concentration in the supernatantwas measured using BCA protein assay reagents (Pierce, 23223 and 23224).A small portion of the supernatant was mixed with sample buffer (Novex,LC2676) and boiled for 3 minutes. The proteins in the supernatant werethen separated by electrophoresis on a 12% SDS-polyacrylamide gel. Equalamounts of total protein were loaded from each cell lysate. Mouseanti-phosphotyrosine (Zymed Laboratories, South San Francisco, AC,03-7720) was used for detection of EGFr tyrosine phosphorylation onWestern blots. ECL western blotting detection reagents (Amersham,Arlington Heights, Ill., RPN2106) and the Hyperfilm ECL (Amersham,Arlington Heights, Ill., RPN1674H) were used for visualizing the signal.The integrated densities of the bands of interest were analyzed using anAGFA Scanner and the Scanalytics OneDscan software (Hewlett Packard,Mountain View, Calif.).

[0422] B. Analysis

[0423] 1. Generation and Characterization of High Affinity NeutralizingFully Human Anti-EGFr MAbs from XenoMouse Strains:

[0424] As described in Example 1, we derived fully human IgG₂κantibodies from transgenic, XenoMouse™, mouse strains throughimmunization with human vulvar epidermoid carcinoma A431 cells. Suchcells express approximately 1×10⁶ EGFr/cell (2, 3 and data not shown).Fusion of B cells from immunized XenoMice with mouse myeloma cellsyielded a panel of hybridomas that secrete human IgG₂κ MAbs specific tothe extracellular domain of human EGFr, as determined by ELISA, BIAcore,Western blots, and flow cytometry analysis (data not shown). The human72 was chosen as the preferred isotype with minimal immune-associatedcytotoxicity against normal EGFr-expressing tissues.

[0425] To identify human MAbs with neutralization activity, purifiedantibodies were evaluated for their ability to inhibit the binding ofEGF and TGFα to human tumor cell lines that express low (colon carcinomaSW948-5×10⁴/cell) or high (A431, or breast adenocarcinomaMDA-MB-468-1×10⁶/cell) levels of EGFr. As positive controls, thecommercially available murine MAbs, 225 and 528, were tested inparallel. A XenoMouse-derived IgG₂κ antibody PK16.3.1, specific forkeyhole lympet hemocyanin (KLH), or a non-specific human myeloma IgG₂Kantibody were used as a negative control. FIG. 45A represents theresults obtained with a subset of the fully human anti-EGFr MAbs testedin these assays. Three of the five human anti-EGFr antibodies shown,E7.6.3, E2.5.1 and E2.3.1, and the mouse anti-EGFr 225 and 528 MAbsblocked the binding of [¹²⁵I]EGF (0.1 nM) to A431 in aconcentration-dependent manner. In contrast, E7.5.2 and E7.8.2 did nothave any effect on EGF binding. The calculated IC₅₀ values (3.0 nM forE7.6.3, 5.6 nM for E2.5.1, 9.1 nM for E2.3.1, 8.8 nM for 225 and 15.2 nMfor 528) suggested E7.6.3 as a potent neutralizing antibody.Furthermore, EGF binding to SW948 cells was also blocked by the humanE7.6.3. and E2.3.1 and by the mouse 225 MAbs (FIG. 45B). The IC₅₀ valuesdetected in studies with SW948 cells were 0.9 nM for E7.6.3, 0.24 nM forE2.3.1, and 0.17 nM for 225. The efficacy of E7.6.3 in neutralizingligand binding was also demonstrated in blocking TGFα binding to A431cells (data not shown). These results indicated that XenoMouse strainsare capable of producing fully human anti-EGFr antibodies whichrecognize different epitopes on the receptor, including those involvedin ligand binding.

[0426] The affinity of the purified E7.6.3 MAb to EGFr was determined tobe 5×10⁻¹¹ M by both solid phase and solution measurements(K_(on)−1.97×10⁶; K_(off)−1.13×10⁻⁴). E7.6.3 exhibits cross-reactivitywith African Green monkey EGFr but not with the mouse EGFr (data notshown). The E7.6.3 hybridoma exhibited significant levels of antibodyproduction that reached a specific productivity rate of 12 pg/cell/dayin serum-free medium growth conditions. Based on its high affinity toEGFr and its potency in blocking EGF/TGFα binding, E7.6.3 MAb wasselected for further evaluation of its efficacy in affecting tumor cellgrowth in vitro and in vivo.

[0427] 2. Antibodies in Accordance with the Present Invention, such asthe E7.6.3 Mab, Inhibit EGF-Mediated Tumor Cell Activation:

[0428] The ability of E7.6.3 to inhibit tumor cell activation wasevaluated by examining its effects on EGF-triggered cellular responsessuch as the tyrosine kinase activity of EGFr, the extracellularacidification rate, and cell proliferation.

[0429] One of the first events following EGF binding to its receptor isthe induction of EGFr tyrosine kinase activity, resulting inautophosphorylation of the receptor (1). As shown in FIG. 46, incubationof human EGF (16 nM) with A431 cells induced the tyrosinephosphorylation of the 170 kDa EGFr. While E7.6.3 did not activate thereceptor tyrosine kinase activity, the antibody blocked EGFr tyrosinephosphorylation in a dose-dependent manner, with a nearly completeinhibition at concentration of 133 nM (antibody: EGFr molar ratio of8:1) (FIG. 46). The E7.6.3 antibody also completely prevented theinternalization of EGF (data not shown). Presumably, the interaction ofE7.6.3 with the receptor led to internalization of the antibody-receptorcomplex but did not activate the receptor tyrosine kinase (FIG. 46).

[0430] Engagement of EGF with its receptor results in cell activation,which is reflected by changes in the extracellular acidification rate.These changes can be detected by the Cytosensor Microphysiometer, apH-sensitive silicon sensor that measures real-time changes in theacidification of the microenvironment surrounding a population ofstimulated cells (22). Using this assay, we examined the effect ofE7.6.3 on EGF-mediated A431 cell activation. As shown in FIG. 47A, theaddition of 1.67 nM EGF to A431 cells induced an immediate increase inthe extracellular acidification rate. No effect was observed when thecells were incubated only with E.7.6.3 antibody at concentrations up to100 nM (not shown). The concurrent addition of E7.6.3 resulted in adose-dependent inhibition of EGF-mediated extracellular acidification(FIG. 47A,B), whereas no effect was detected with the isotype matchedcontrol antibody PK16.3.1 (FIG. 47B).

[0431] Lastly, we examined the effect of E7.6.3 on the in vitroproliferation of the EGFr-expressing tumor cell lines A431 andMDA-MB-468, again in comparison to the mouse anti-EGFr antibodies. Bothcell lines, expressing high levels of EGFr, have been shown to secreteTGFα and to be growth inhibited by the addition of exogenous EGF at nMconcentrations (24,25). Therefore, the experiments using these two celllines were carried out in the absence of exogenous EGF. E7.6.3 inhibitedthe growth of A431 cells in a dose-dependent manner, with a maximalinhibition of 60%, a level similar to that obtained with the mouseantibody 225 and higher than that observed for the 528 antibody (FIG.48A). The control antibody did not have any effect on cell proliferation(FIG. 48A). The calculated IC₅₀ values for E7.6.3 (0.125 nM), 225 (0.48nM) or 528 (0.66 nM) antibodies, indicated E7.6.3 efficacy in inhibitingA431 cell proliferation (FIG. 48A). A similar level of growth inhibitionby E7.6.3 was observed with MDA-MB-468 cells (FIG. 48B). Since noexogenous EGF was added to the culture, these results indicate theability of the human antibody to block autocrine growth stimulation andthus to inhibit EGF/TGFα-induced tumor cell activation. In experimentscarried out with SW948 cells, 10 nM of E7.6.3 MAb blocked completely theproliferation of the cells (data not shown).

[0432] 3. Antibodies in Accordance with the Invention, such as theE7.6.3 Mab, Prevents Human Tutor Formation in Mice:

[0433] To examine the effect of E7.6.3 on tumor cell growth in vivo, theantibody was first evaluated for its ability to prevent the formation ofA431 tumor xenografts in athymic mice. A431 cells (5×10⁶/mouse) wereinjected subcutaneously (s.c.) into mice in conjunction withintraperitoneal (i.p.) administration of either PBS (group 1), 1 mg ofthe control antibody PK16.3.1 (group 2), or 0.2 mg or 1 mg of E7.6.3(groups 3 and 4). The antibody administration was repeated twice a weekover three weeks, for a total dose of 1.2 mg (group 3) or 6 mg (groups 2and 4). As shown in FIG. 52, all mice treated with either PBS or thecontrol antibody developed tumors by day 10 after inoculation and wereeuthanized at day 30 due to the large size of the tumorss. In contrast,none of the mice treated with E7.6.3 antibody developed tumors for morethan 8 months following the last antibody injection. The data indicatedthat E7.6.3 prevented the formation of A431 xenografts, probably byexerting its neutralization activity at the initial phase of the tumorcell proliferation.

[0434] 4. Antibodies in Accordance with the Invention, such as theE7.6.3 Mab, Eradicate Large Human Tumor Xenografts in Mice:

[0435] The effect of E7.6.3 on the growth of established tumors wasexamined on A431 tumor xenografts that reached a size of 0.13 to 1.2 cm³(calculated as length x width x height×π/6). Initially, mice bearing0.13-0.25 cm³-sized tumors were treated i.p. with 1 mg of either E7.6.3Mab or the human myeloma IgG₂κ control antibody, twice a week over threeweeks. As shown in FIG. 49 and in FIG. 53, the tumors in untreated miceor mice treated with the control antibody continued their aggressivegrowth to reach the size of 3 cm³ by day 30, at which point the micewere euthanized. In contrast, treatment with E7.6.3, not only arrestedfurther growth of the tumors but also caused continuous tumor regressionthat resulted in a complete tumor eradication in all mice treated (FIGS.49 and 53). No recurring tumors were detected for more than 250 days inany of the mice that were monitored, demonstrating a long-lasting effectof the antibody and its ability to completely eliminate all tumor cells.

[0436] We next evaluated the potency of E7.6.3 antibody to treat largeestablished tumor xenografts. Mice bearing 0.13, 0.56, 0.73 or 1.2cm³-sized A431 tumors were treated i.p. with 1 mg E7.6.3 twice a week,over three weeks, initiated on day 7, 11, 15 or 18, respectively. Asdemonstrated in FIG. 49, E7.6.3 caused a profound tumor regression inall the treated mice regardless of their initial tumor size, even withtumors as large as 1.2 cm³. Furthermore, this treatment led to acomplete disappearance of the tumors (FIG. 49) and no tumor recurrencein any of the mouse groups for 210 days after the last antibodyinjection (data not shown).

[0437] As summarized in FIG. 53, the antibody effect appears to bedose-dependent with a total dose of 3 mg leading to a nearly completetumor eradication and a total dose of 0.6 mg eliminating 65% of theestablished A431 xenografts.

[0438] To compare the anti-tumor activity of E7.6.3 to that of the mouse225 antibody, which was reported to affect the growth of establishedA431 tumors but not cause their elimination (12,13), we used suboptimalE7.6.3 doses (0.05 mg and 0.2 mg, given twice a week for three weeks)that also caused primarily tumor regression in A431 xenografts. At theseantibody doses, there was a significant difference between in theability of E7.6.3 and 225 to arrest the growth of A431 xenografts (FIG.49C).

[0439] E7.6.3 was also shown to be efficacious in inhibiting the growthof the breast carcinoma MDA-MB-468 xenografts (FIG. 50A). Treatment of0.2 cm³ tumor-bearing mice with 2 mg antibody once a week over 2 weeksled to a complete arrest of the tumor growth. The fact that there was noapparent change in the residual nodules for 120 days after the lastantibody administration, strongly suggests that the antibody effectivelyeradicated these tumors.

[0440] A similar anti-tumor activity of E7.6.3 was observed when theantibody was given via different administration routes (FIG. 50B).Administration of 0.5 mg E7.6.3 into mice carrying 0.15 cm³-sized A431xenografts twice a week over three weeks by either i.p., s.c., i.v., ori.m route all caused complete tumor eradication.

[0441] The elimination of all tumor cells by E7.6.3 was furthersupported by the histopathological analysis of the small residualnodules observed in some of the A431 xenograft-bearing mice that weretreated with the lower antibody doses. Biopsies taken from these nodulesat day 79 were shown to contain a thin fibrovascular capsule lined bynecrotic cells with its center filled with keratinic and calcifieddebris (FIG. 51A). There was no evidence of neoplastic cells, which werereadily detected in tumors taken from mice treated with PBS or controlantibody (FIG. 51B). A mild inflammatory infiltration of neutrophils,lymphocytes, plasma cells, macrophages and multinucleated giant cellssurrounded the capsule. Taken together with the long lasting inhibitoryeffect, the data strongly suggest complete tumor cell eradication byE7.6.3 antibody.

[0442] C. Discussion

[0443] Utilization of XenoMouse animals for the production of humanantibodies specific to the human EGFr yielded the fully human IgG₂κ Mab,E7.6.3, characterized by high affinity and strong neutralizationactivity. Its demonstrated efficacy in eradicating large establishedhuman tumor xenografts without concomitant chemotherapy stronglysuggests it as a suitable candidate for antibody monotherapy in patientswith EGFr-expressing tumors.

[0444] E7.6.3 exhibited strong efficacy in blocking the binding of EGFand TGFα to EGFr on the surface of different human carcinoma cell lines,including those that express high levels of receptors (FIG. 45). Thecomplete inhibition of ligand binding to the receptors on A431 and SW948cells resulted in an abolishment of the signaling events triggered byEGF or TGFα, including EGFr autophosphorylation and internalization,increased extracellular acidification rate, and cell proliferation. Ourresults indicate that E7.6.3 can block ligand-induced cell activationand that E7.6.3 does not act as an agonist to trigger cellular responsesin EGFr-expressing tumors (FIGS. 2,3).

[0445] The anti-tumor activity of E7.6.3 was examined in multiplexenograft mouse experiments, in which the effects of various antibodydoses on different sizes of tumors were established (FIGS. 5,6). Theresults obtained from these studies demonstrated the unique anti-tumorproperties of E7.6.3 MAb as compared to the other reported anti-EGFrantibodies. E7.6.3 not only arrested the growth of human tumorxenografts but also completely eradicated established tumors by itself,without the need for concomitant chemotherapy. Tumor eradication of A431xenografts was achieved in nearly all mice treated with total doses aslow as 3 mg, administered over the course of 3 weeks, and a total doseof 0.6 mg led to tumor elimination in 65% of the mice (FIGS. 5, 6B,Table 2). In comparison, 8 mg of 225 and 10 mg of 528 antibodies, givenover 4 and 10 weeks, respectively, had only a limited effect on A431tumors and required the co-administration of chemotherapeutic drugs toachieve the elimination of the tumors (12,13). A direct comparisonbetween E7.6.3 and 225 MAbs at low doses demonstrated E7.6.3 as a morepotent antibody in regressing established A431 tumors and arrestingtheir growth (FIG. 49C). The chimeric C225 MAb, which was reported toacquire higher affinity to EGFr and enhanced in vivo anti-tumoractivities, achieved complete A431 tumor eradication at a total dose of10 mg, given over 5 weeks, whereas total doses of 2.5 and 5 mg led toonly 14% and 57% of tumor-free mice (14). The potent anti-tumor activityof E7.6.3 was further validated by its ability, at a 6 mg total dose, tocompletely eliminate established tumors as large as 1.2 cm³ in all micetreated.

[0446] This anti-tumor potency of E7.6.3 is likely to originateprimarily from the antibody's intrinsic activity as its human γ2 isotypewas shown to minimally engage the immune system-derived effectorfunctions, such as cell-mediated cytotoxicity or complement-dependentcytolysis. In comparison, the anti-tumor activities of the rat ICR62,mouse 528 or chimeric C225 antibodies were suggested to reflect theparticipation of the host immune effector functions recruited by therespective rodent γ2b or human γ1 isotypes (2,4,6,26).

[0447] The molecular mechanism(s) underlying the potent anti-tumoractivity of E7.6.3 appear to be contributed to by several factors,including, (i) the antibody's ability to block ligand-triggered growthand survival signals and (ii) the effects that the antibody may exert onthe cell upon its interaction with the receptor. The potency of E7.6.3can be attributed, at least in part, to the high affinity (5×10⁻¹¹ M)that the antibody exhibits to human EGFr, higher than the affinityreported for other anti-EGFr MAbs (12,14). With its high affinity,E7.6.3 can inhibit or dissociate the ligand binding to the receptorsvery effectively, thus depriving the cells completely from receivingessential growth and survival stimuli. Like other anti-EGFr antibodies(2,4,6), E7.6.3 MAb does not act as an agonist and does not activatecells upon binding to the receptor. The difference in efficacy betweenE7.6.3 and the other antibodies tested in xenograft mouse models canalso be attributed to a unique E7.6.3 binding epitope on EGFr that canmediate a stronger neutralization effect or induce cell cytotoxicity.The latter factor is supported by the ability of E7.6.3 to eradicatewell established human xenografts, as large as 1.2 cm³. The mechanismbehind the in vivo cytocidal effects of E7.6.3 may involve the inductionof either programmed cell death, differentiation of the tumor cells, ormodulation of expression of angiogenesis factors, mechanisms that wereshown to be triggered by antibodies in cultured cells (27-31). Differentmechanisms may account for the antibody effect on different tumors andin some cases probably more than one mechanism contributes to theanti-tumor activity.

[0448] The potency of E7.6.3 in eradicating well established tumorssuggests that this antibody can provide effective therapy to tumors thatrequire EGFr activation for their continuous progression and survival.As E7.6.3 does not require the presence of chemotherapy to exert itanti-tumor activity, the antibody could be applied to variousEGFr-expressing human solid tumors. Furthermore, being a fully humanantibody, E7.6.3 is expected to have a long serum half life and minimalimmunogenicity with repeated administration, including in allimmunocompetent patients. In addition, bearing a human γ2 constantregion that interacts poorly with the effector arm of the immune system,E7.6.3 MAb may not induce cytotoxic effects on normal EGFr-expressingtissues such as liver and skin.

[0449] Utilization of Mabs directed to growth factor receptors as cancertherapeutics has been validated recently by the tumor responses obtainedfrom clinical trials with Herceptin™, the humanized anti-HER2 antibody,in patients with HER2 overexpressing metastatic breast cancer (32, 33).The potent in vivo anti-tumor activity of E7.6.3, as demonstrated inthis report, suggests it as a good candidate for assessing thetherapeutic potential of anti-EGFr therapy in EGFr-expressing humantumors.

EXAMPLE 12 Valency of Human Anti-EGF-r Antibodies

[0450] Valency has been indicated to play a role in certain inconnection with certain antibodies that bind to EGFr. For example, Masuiet al. Cancer Research 46:5592-5598 (1986) conducted certaininvestigations related to the 528 and 225 antibodies and postulated thatvalency of the antibodies could play a role in the mechanism of actionof the antibodies. It was unclear for the paper, however, whether theeffects observed were based upon valency or upon a form of complementfixation/cytotoxicity. More recently, investigations have highlightedthat valency may play an important role in connection with either thefacilitation or prevention of certain dimerization events in connectionwith certain cell-surface receptors in oncogenesis. See, for example,Chanty A. J. Biol. Chem. 270:3068-3073 (1995); Wallasch et al. EMBO J.14:4267-4275 (1995); Earp et al. Breast Cancer Research Treatment35:115-132 (1995); and Zhang et al. J Biol. Chem. 271:3884-3890 (1996).

[0451] Based upon the results observed in FIG. 45A, we observed asignificantly different slope between the inhibition curve for E7.6.3antibody and that for the 225 antibody. Such difference may be basedupon valency. Accordingly, in order to further investigate the valencyof the E7.6.3 antibody, we plan to conduct saturation studies on theE7.6.3 antibody (or other antibodies in accordance with the invention)as compared to the 225 and/or 528 antibody. In the studies, the testantibodies will be iodinated with radioactive iodine using conventionaltechniques and varying quantities of the test antibodies (untilsaturation) will be introduced to wells containing known numbers of EGFreceptors. Bound antibodies will be determined using counting.

[0452] A difference in valency could be indicative of a role ofantibodies in accordance with the invention in effecting dimerization ofEGF receptor.

EXAMPLE 13 Human Clinical Trials for the Treatment and Diagnosis ofHuman Carcinomas through use of Human Anti-EGF-r Antibodies in vivo

[0453] Introduction

[0454] Antibodies in accordance with the present invention are indicatedin the treatment of certain solid tumors. Based upon a number offactors, including EGF-r expression levels, among others, the followingtumor types appear to present preferred indications: breast, ovarian,colon, prostate, bladder and non-small cell lung cancer. In connectionwith each of these indications, three clinical pathways appear to offerdistinct potentials for clinical success:

[0455] Adjunctive therapy: In adjunctive therapy, patients would betreated with antibodies in accordance with the present invention incombination with a chemotherapeutic or antineoplastic agent and/orradiation therapy. The primary targets listed above will be treatedunder protocol by the addition of antibodies of the invention tostandard first and second line therapy. Protocol designs will addresseffectiveness as assessed by reduction in tumor mass as well as theability to reduce usual doses of standard chemotherapy. These dosagereductions will allow additional and/or prolonged therapy by reducingdose-related toxicity of the chemotherapeutic agent. Prior artanti-EGF-r antibodies have been, or are being, utilized in severaladjunctive clinical trials in combination with the chemotherapeutic orantineoplastic agents adriamycin (C225: advanced prostrate carcinoma),cisplatin (C225: advanced head and neck and lung carcinomas), taxol(C225: breast cancer), and doxorubicin (C225: preclinical).

[0456] Monotherapy: In connection with the use of the antibodies inaccordance with the present invention in monotherapy of tumors, theantibodies will be adminstered to patients without a chemotherapeutic orantineoplastic agent. Preclinical results generated through use ofantibodies in accordance with the present invention and discussed hereinhave demonstrated similar results with both adjunctive therapy and/or asa stand-alone therapy. Moreover, monotherapy has apparently beenconducted clinically in end stage cancer patients with extensivemetastatic disease. Patients appeared to show some diseasestabilization. Id. Trials will be designed to demonstrate an effect inrefractory patients with (cancer) tumor.

[0457] Imaging Agent: Through binding a radionuclide (e.g., yttrium(⁹⁰Y)) to antibodies in accordance with the present invention, it isexpected that radiolabeled antibodies in accordance with the presentinvention can be utilized as a diagnostic, imaging agent. In such arole, antibodies of the invention will localize to both solid tumors, aswell as, metastatic lesions of cells expressing the EGF receptor. Inconnection with the use of the antibodies of the invention as imagingagents, the antibodies can be used in assisting surgical treatment ofsolid tumors, as both a pre-surgical screen as well as a post operativefollow to determine what tumor remain and/or returns. An (¹¹¹IN)-C225antibody has been used as an imaging agent in a Phase I human clinicaltrial in patients having unresectable squamous cell lung carcinomas.Divgi et al. J. Natl. Cancer Inst. 83:97-104 (1991). Patients werefollowed with standard anterior and posterior gamma camera. Preliminarydata indicated that all primary lesions and large metastatic lestionswere identified, while only one-half of small metastatic lesions (under1 cm) were detected.

[0458] Dose and Route of Administration

[0459] While specific dosing for antibodies in accordance with theinvention has not yet been determined, certain dosing considerations canbe determined through comparison with the similar product (ImClone C225)that is in the clinic. The C225 antibody is typically being administeredwith doses in the range of 5 to 400 mg/m², with the lower doses usedonly in connection with the safety studies. Antibodies in accordancewith the invention have a one-log higher affinity than the C225antibody. Further, antibodies in accordance with the present inventionare fully human antibodies, as compared to the chimeric nature of theC225 antibody and, thus, antibody clearance would be expected to beslower. Accordingly, we would expect that dosing in patients withantibodies in accordance with the invention can be lower, perhaps in therange of 50 to 300 mg/M², and still remain efficacious. Dosing in mg/m²,as opposed to the conventional measurement of dose in mg/kg, is ameasurement based on surface area and is a convenient dosing measurementthat is designed to include patients of all sizes from infants toadults.

[0460] Three distinct delivery approaches are expected to be useful fordelivery of the antibodies in accordance with the invention.Conventional intravenous delivery will presumably be the standarddelivery technique for the majority of tumors. However, in connectionwith tumors in the peritoneal cavity, such as tumors of the ovaries,biliary duct, other ducts, and the like, intraperitoneal administrationmay prove favorable for obtaining high dose of antibody at the tumor andto minimize antibody clearance. In a similar manner certain solid tumorspossess vasculature that is appropriate for regional perfusion. Regionalperfusion will allow the obtention of a high dose of the antibody at thesite of a tumor and will minimize short term clearance of the antibody.

[0461] Clinical Development Plan (CDP)

[0462] Overview: The CDP will follow and develop treatments ofanti-EGF-r antibodies in accordance with the invention in connectionwith adjunctive therapy, monotherapy, and as an imaging agent. Trialswill be initially utilized to demonstrate safety and will thereafter beutilized to address efficacy in repeat doses. Trails will be open labelcomparing standard chemotherapy with standard therapy plus antibodies inaccordance with the invention. As will be appreciated, one criteria thatcan be utilized in connection with enrollment of patients can be EGF-rexpression levels of patient tumors as determined in biopsy.

[0463] As with any protein or antibody infusion based therapeutic,safety concerns are related primarily to (i) cytokine release syndrome,i.e., hypotension, fever, shaking, chills, (ii) the development of animmunogenic response to the material (i.e., development of humanantibodies by the patient to the human antibody therapeutic, or HAHAresponse), and (iii) toxicity to normal cells that express the EGFreceptor, e.g., hepatocytes which express EGF-r. Standard tests andfollow up will be utilized to monitor each of these safety concerns. Inparticular, liver function will be monitored frequently during clinicaltrails in order to assess damage to the liver, if any.

[0464] Human Clinical Trial:

[0465] Adjunctive Therapy with Human Anti-EGF-r Antibody andChemotherapeutic Agent

[0466] A phase I human clinical trial will be initiated to assess thesafety of six intravenous doses of a human anti-EGF-r antibody inaccordance with the invention in connection with the treatment of asolid tumor, e.g., breast cancer. In the study, the safety of singledoses of antibodies in accordance with the invention when utilized as anadjunctive therapy to an antineoplastic or chemotherapeutic agent, suchas cisplatin, topotecan, doxorubicin, adriamycin, taxol, or the like,will be assessed. The trial design will include delivery of six, singledoses of an antibody in accordance with the invention with dosage ofantibody escalating from approximately about 25 mg/m² to about 275 mg/m²over the course of the treatment in accordance with the followingschedule: Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 Mab Dose 25 75 125 175225 275 mg/m² mg/m² mg/m² mg/m² mg/m² mg/m² Chemotherapy + + + + + +(standard dose)

[0467] Patients will be closely followed for one-week following eachadministration of antibody and chemotherapy. In particular, patientswill be assessed for the safety concerns mentioned above: (i) cytokinerelease syndrome, i.e., hypotension, fever, shaking, chills, (ii) thedevelopment of an immunogenic response to the material (i.e.,development of human antibodies by the patient to the human antibodytherapeutic, or HAHA response), and (iii) toxicity to normal cells thatexpress the EGF receptor, e.g., hepatocytes which express EGF-r.Standard tests and follow up will be utilized to monitor each of thesesafety concerns. In particular, liver function will be monitoredfrequently during clinical trails in order to assess damage to theliver, if any.

[0468] Patients will also be assessed for clinical outcome, andparticularly reduction in tumor mass as evidenced by MRI or otherimaging.

[0469] Assuming demonstration of safety and an indication of efficacy,Phase II trials would likely be initiated to further explore theefficacy and determine optimum dosing.

[0470] Human Clinical Trial:

[0471] Monotherapy with Human Anti-EGF-r Antibody

[0472] Assuming that the antibodies in accordance with the presentinvention appear safe in connection with the above-discussed adjunctivetrial, a human clinical trial to assess the efficacy and optimum dosingfor monotherapy. Such trial could be accomplished, and would entail thesame safety and outcome analyses, to the above-described adjunctivetrial with the exception being that patients will not receivechemotherapy concurrently with the receipt of doses of antibodies inaccordance with the invention.

[0473] Human Clinical Trial:

[0474] Diagnostic Imaging with Anti-EGF-r Antibody

[0475] Once again, assuming that the adjunctive therapy discussed aboveappears safe within the safety criteria discussed above, a humanclinical trial can be conducted concerning the use of antibodies inaccordance with the present invention as a diagnostic imaging agent. Itis expected that the protocol would be designed in a substantiallysimilar manner to that described in Divgi et al. J. Natl. Cancer Inst.83:97-104 (1991).

EXAMPLE 14 Additional Characterization of Antibodies in Accordance withthe Invention

[0476] In order to further characterize antibodies in accordance withthe invention, we conducted a number of additional in vivo assays. Inaddition to the assays discussed above (i.e., in connection with Example10), certain of such assays were conducted in accordance with thefollowing Materials and Methods:

[0477] A. Materials and Methods

[0478] Tumor Xenograft Mouse Models:

[0479] In this experiment, we evaluated the following tumor cell lines:three human pancreatic carcinoma cell lines (HPAC, BxPC-3, HS766T (eachobtained from the ATCC: HPAC (ATCC, CRL-2219), BxPC-3 (ATCC, CRL-1687),HS766T (ATCC, HTB-134))), the human kidney carcinoma cell line, SK-RC-29(obtained from the Memorial Sloan-Kettering Cancer Center, NY, N.Y.),and the human colon carcinoma cell line, SW707 (obtained from theDeutsches Krebsforschungzentrum (German Cancer Research Institute),Heidelberg, Del.). Male BALB/c-nu/nu mice (6-8 weeks of age) wereinjected s.c. with 5×10⁶ A431 or HPAC, BxPC-3, HS766T, SK-RC-29, orSW707 cells in 100 μl PBS. Tumors sizes were measured twice a week witha vernier caliper and tumor volume was calculated as the product oflength×width×height×π/6. Mice with established tumors were randomlydivided into treatment groups. Mice received antibody treatment twice aweek over three consecutive after tumor establishment. Tumors weremeasured twice a week.

[0480] B. Analysis

[0481] 1. Antibodies in Accordance with the Invention, such as theE7.6.3 Mab, Eradicate Human Tumor Xenografts in Mice.

[0482] To understand the underlying mechanism of the in vivo anti-tumoractivity of E7.6.3, mice bearing A431 tumors were treated i.p with. twodifferent human anti-EGF-r Mabs, E7.6.3 or E7.5.2 (1 mg/mouse). Althoughboth Mabs bind human EGF-r, only E7.6.3 blocks the binding of EGF orTGFα to EGF-r while E7.5.2 does not. As shown in Figure A, the tumorswere completely eradicated on day 60 by the neutralizing antibody E7.6.3while E7.5.2 had almost no effect on tumor growth as compared to thecontrol. The data suggest that the in vivo anti-tumor activity ofanti-EGF-r antibody requires the blockade of EGF-r binding sites.

[0483] 2. Antibodies in Accordance with the Invention, such as theE7.6.3 Mab, does not Affect EGF-r Negative Human Tumor Xenografts inMice.

[0484] To understand the underlying mechanism of the in vivo anti-tumoractivity of E7.6.3, human colon tumor cells SW707 which do not expressEGF-r were injected s.c. into nude mice. Mice bearing established SW707tumors were treated i.p with. 1 mg of human anti-EGF-r Mab, E7.6.3.Control mice received no treatment. As shown in Figure F, treatment withE7.6.3 at 1 mg twice a week for three weeks failed to affect the growthof SW707 tumor indicating that the in vivo anti-tumor activity ofanti-EGF-r antibody is antigen-specific.

[0485] 3. Antibodies in Accordance with the Invention, such as theE7.6.3 Mab, Inhibits the Growth of Multiple Human Tumor Xenografts inMice.

[0486] The effect of E7.6.3 on the growth of multiple different humantumors was examined in xenograft mice. Three human pancreatic tumor celllines, HPAC, BxPC-3 or HS766T, or a human renal tumor SK-RC-29 wereinjected into nude mice. The mice bearing the established tumors weretreated i.p. with 1 mg of E.7.6.3 twice a week for three weeks. E7.6.3treatment resulted in growth inhibition of HPAC during and 12 days afterantibody treatment. Nevertheless, the inhibitory effect disappeared 12days after termination of the treatment suggesting that for some tumorsa sustained inhibition of tumor growth may require a prolonged antibodytreatment. In contrast, administration of E7.6.3 twice a week for threeweeks led to a significant and extended tumor growth arrest of BxPC-3,HS766T and SK-RC-29. Since the expression level of EGF-r on HPAC is muchlower than BxPC-3, HS766T and SK-RC-29 (data not shown), it appearspossible that tumors that have high levels of EGF-r expressed on theircell surfaces respond preferentially to the anti-EGFr antibodytreatment.

EXAMPLE 15 Additional Characterization of Antibodies in Accordance withthe Invention

[0487] Example 10 presented information related to the inhibition ofEGF-r phosphorylation and preliminary data related to theinternalization of the EGF-r by cells. Further to the detaileddiscussion in Example 10 related to additional characterization of theantibodies in accordance with the present invention, we havedemonstrated additional activities that appear to be important to theactivity of the E7.6.3 antibody of the invention. the

[0488] Materials and Methods

[0489] Internalization of EGFr

[0490] In order to study the effect anti-EGF-r antibodies oninternalization of EGF-r, confluent A431 cells in 24 well plates werewashed and incubated with 10 ng/ml of ¹²⁵I-EGF or 200 ng/ml of¹²⁵I-E7.6.3 at 4° C. for 90 min, and then incubated at 37° C. fordifferent times to allow internalization. The plates were then placed onice and washed. Surface-bound ligand was collected by two washes with0.5 M acetic acid, 150 mM NaCI, and the cells were lysed with 1 ml of 1NNaOH for 30 min at 37° C. The radioactivity in the acetic acid and NaOHwas counted in β-counter. See FIG. 80.

[0491] EGF and EGFr Degradation

[0492] In order to study the effect of anti-EGF-r antibodies ondegradation of EGF-r, 70% confluent A431 cells were labeled with³⁵S-Methionine in methionine free medium containing 10% FBS for 16 hrs.After labeling, the cells were washed with PBS and incubated with serumfree DMEM/F12 medium for 1 hr. The cells were then treated with 16 nM ofEGF and 133 nM of either the E7.6.3 antibody, 225 antibody for 30 min,or a negative control. As controls, either K221 (a human IgG2 anti-IL-8antibody) or a murine anti-IgG1 antibody were used. After the 30-minincubation with the antibody, the cells were washed three times withcold PBS and scraped into 0.5 ml of lysis buffer (10 mM Tris, 150 mMNaCl, 5 mM EDTA, 1% triton-100, 0.1 mg/ml PMSF, 1 μg/ml aprotinin, 1μg/ml leupeptin, 50 mM NaF, 40 mM β-glycerol phosphate, 10 mMpyrophosphate, 10 mM Hepes pH 7.3, and 1 mM sodium orthovanadate). After30 minutes of incubation on ice, the lysate was centrifuged at 10,000rpm for 5 minutes in an Eppendorf centrifuge at 4° C. 100 μl of lysatewas immunoprecipitated using the E7.5.2 (discussed above, anon-neutralizing human anti-EGFr antibody) using protein A Sepharosebeads. The protein A Sepharose-E7.5.2-protein (in lysate) complex werewashed three times, mixed with 2× SDS sample buffer and boiled for 4min. The proteins in samples were separated by electrophoresis on a 10%SDS-polyacrylamide gel. The gels were then fixed and dried beforeexposing to a film. See FIGS. 81 and 82.

[0493] EGF-r Threonine Phosphorylation

[0494] In order to study the effects of anti-EGF-r antibodies onthreonine phosphorylation of EGF-r, 70% confluent A431 cells werelabeled with ³⁵S-Methionine in methionine free medium containing 10% FBSfor 16 hrs. After labeling, the cells were washed with PBS and incubatedwith serum free DMEM/F12 medium for 1 hr. The cells were then treatedwith or without EGF (5 or 10 nM) in the absence or presence of theE7.6.3 antibody or the 225 antibody (200 nM) for 30 minutes. The K2.2.1antibody was used as a negative control. After the 30-min incubation,the cells were washed three times with cold PBS and scraped into 0.5 mlof lysis buffer (10 mM Tris, 150 mM NaCl, 5 mM EDTA, 1% triton-100, 0.1mg/ml PMSF, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 50 mM NaF, 40 mMβ-glycerol phasphate, 10 mM pyrophasphate, 10 mM Hepes pH 7.3, and 1 mMsodium orthovanadate). After 30 minutes of incubation on ice, the lysatewas centrifuged at 10,000 rpm for 5 minutes in an Eppendorf centrifugeat 4° C. Two sets of 100 μl of lysate were prepared andimmunoprecipitated with the E7.5.2 antibody or rabbitanti-phosphothreonine antibody (available from Zymed, South SanFrancisco, Calif.) using protein A Sepharose beads. The protein ASepharose-antibody-protein (in lysate) complex were washed three times,mixed with 2× SDS sample buffer and boiled for 4 min. The proteins insamples were separated by electrophoresis on a 10% SDS-polyacrylamidegel. The gels were then fixed and dried before exposing to a film (filmexposure and visualization was accomplished as described in Example 10).See FIG. 83.

[0495] In another experiment, in an effort to reduce the autocrineproduction of EGF by cells, we used 70% confluent A431 cells werepre-incubated at a low concentration of fetal bovine serum (0.5%)overnight in 37° C. The cells were then treated with 16 nM EGF in thepresence or absence of different concentrations of E7.6.3 MAb for 30minutes at 37° C. After the 30-min incubation, the cells were washedthree times with cold PBS and scraped into 0.5 ml of lysis buffer (10 mMTris, 150 mM NaCl, 5 mM EDTA, 1% Triton-100, 0.1 mg/ml PMSF, 1 μg/mlaprotinin, 1 μg/ml leupeptin and 1 mM sodium orthovanadate). After 30minutes of incubation on ice, the lysate was centrifuged at 10,000 rpmfor 5 minutes in an Eppendorf centrifuge at 4° C. Two sets of 100 μl oflysate were prepared and immunoprecipitated with the E7.5.2 antibody orrabbit anti-phosphothreonine antibody (available from Zymed, South SanFrancisco, Calif.) using protein A Sepharose beads. The protein ASepharose-antibody-protein (in lysate) complex were washed three times,mixed with 2× SDS sample buffer and boiled for 4 min. The proteins insamples were separated by electrophoresis on a 10% SDS-polyacrylamidegel. The gels were then fixed and dried before exposing to a film (filmexposure and visualization was accomplished as described in Example 10).See FIG. 84.

[0496] Vascular Endothelial Cell Growth Factor (VEGF) Production inTumor Cells

[0497] In order to study the effects of anti-EGF-r antibodies on theupregulation of VEGF production in tumor cells, 70% confluent A431 cellsin 24 well plates were washed with PBS and re-feed with fresh medium.The cells were treated with or without various antibodies as indicatedand incubated for 48 or 96 hrs. At the end of culture, medium wascollected and VEGF concentration in the medium was determined usingELISA kit (VEGF ELISA kit purchased from R&D, Minneopolis, Minn.). E752and K221 were used as negative controls. Four individual experiments arepresented. See FIG. 85.

[0498] VEGF Production in Endothelial Cells

[0499] In order to study the effects of anti-EGF-r antibodies on theupregulation of VEGF production in endotelial cells, 70% confluentECV304 cells (ATCC: CRL-1998) in 24 well plates were washed with PBS andre-fed with fresh medium. The cells were treated with or without EGF inthe presence or absence of various antibodies as indicated and incubatedfor 24 hrs. At the end of culture, medium was collected and VEGFconcentration in the medium was determined using ELISA kit (VEGF ELISAkit purchased from R&D, Minneopolis, MN). K221 was used as negativecontrols. See FIG. 86.

[0500] Discussion

[0501] The above experiments provide important additional informationabout antibodies in accordance with the invention. For example, in FIG.80 demonstrates that EGF-r is internalized after binding to either EGF(panel A) or the E7.6.3 antibody (panel B). Thereafter, a questionarises is whether EGF-r is then degraded once internalized. FIG. 81demonstrates that E7.6.3 is not degraded (panel B) and that thedegradation of EGF provides a positive control (panel A). With respectto the degradation of EGF-r, FIG. 82 provides a series ofimmunoprecipitation blots that compare the effects of various antibodieson EGF-r degradation. As will be observed, when EGF binds to EGF-r,EGF-r degradation is induced. Panel A. Also, the 225 antibody inducedEGF-r degradation. Treatment with either E7.6.3 or the non-specific K221antibodies did not induce degradation of the receptor. In panel B,similar results are observed with the additional demonstration that themurine IgG1 did not induce degradation. Since the 225 antibody is amurine IgG1, the murine IgG1 acts as a negative control for 225 andindicates that the induction of degradation by 225 is specific. In panelC, EGF induced degradation of the receptor is shown. The resultsdemonstrate that the E7.6.3 antibody completely inhibit the effect ofEGF induced EGF-r degradation. In contrast, the 225 antibody did notinhibit the EGF induced EGF-r degradation.

[0502] From the data related EGF-r tyrosine phosphorylation, discussedin Example 10, where both the E7.6.3 and 225 antibodies inhibited EGF-rtyrosine phosphorylation and the difference between E7.6.3 and 225 withrespect to the effect on EGF-r degradation, we sought to determine ifthere were additional phosphorylation differences related to thereceptor. Accordingly, an experiment was conducted to view threoninephosphorylation of EGF-r. In FIG. 83, panel A shows immunoprecipitationof EGF-r by the E7.5.2 antibody, indicating that the quantity of EGF-rwas the same. In panel B, shows immunoprecipitation by the rabbitanti-phosphothreonine antibody. As will be observed, significantthreonine phosphorylation of the receptor is preserved by treatment withthe E7.6.3 antibody. In contrast, a majority of the threoninephosphorylation is vitiated in treatement with the 225 antibody. Aninteresting additional band of threonine phosphorylation was seen atabout 63 KD in the E7.6.3 treated cells.

[0503] Such additional band was further explored in connection with theresults presented in FIG. 84. In this experiment, in an effort to reducethe autocrine production of EGF by cells, we raised the cells withalmost no FBS. In the control group, spontaneous threoninephosphorylation is seen in a band at about 63 KD. EGF dramaticallyreduced such phosphorylation. EGF in combination with severalneutralizing anti-EGF-r antibodies, including E7.6.3, partially restoredsuch phosphorylation.

[0504] We also studied the production of vascular endothelial cellgrowth factor (VEGF) in tumor (A431) cells. VEGF is an importantmodulator of growth of endothelial cells and a potent angiogenic factor.It is believed to be important to new blood vessel formation in tumors.E.g., Liu and Ellis Pathobiology 66:247-252 (1998). In FIG. 85, thelevels of VEGF production were examined and E7.6.3 significantly (>70%)inhibited VEGF production. In contrast, the 225 antibody inhibited VEGFproduction by much less than 50%, more accurately around 25%. Theantibodies E752 and K221 were used as negative controls and they do notinhibit VEGF production.

[0505] In addition to the tumor cell work, we have completed preliminaryexperiments with respect to vascular endothelial cells. In addition tobeing recruited to sites of angiogenesis, such cells express EGF-r ontheir surfaces. Wilson and Lloyd Invest. Opthamol. & Visual Science32:2747-2756 (1991). In FIG. 86, based on the data from 24 hourincubation, it will be observed that when such endothelial cells arestimulated with EGF, the VEGF production is increased. However,treatement with the E763 antibody inhibits the VEGF production by atleast 40%. The 225 antibody on the other hand inhibits VEGF productionby significantly less (20%).

[0506] A table summarizing certain of the above demonstratedsimilarities and differences between the E763 antibody and the 225antibody is provided below: TABLE II Characteristic E7.6.3 225Inhibition of Tyrosine Phosphorylation + + Internalization of EGF-r + +Inhibition of EGF-r Degradation EGF-r + − EGF-induced + −Thr-Phosphorylation EGF-r + +/− 63 KD Protein + − Inhibition of VEGFProduction Tumor Cells >50% <50% Endothelial Cells >40% <40%

[0507] The data presented herein demonstrates the significant functionaldifferences between the E763 and the 225 antibodies. In particular, theresults with respect to VEGF production and the potential inhibition ofendothelial cell proliferation within a tumor during the growth of atumor lead naturally to a study of the downstream conditions of tumorgrowth. For example, it is expected that the E763 antibody may act toinhibit tumor cells and cells that depend on VEGF for their growth suchas endothelial cells. In addition, other downstream molecules such asthe VEGF receptor in endothelial cells and the activity of tPA in tumorcells and endothelial cells are expected to be affected by antibodies inaccordance with the invention. Accordingly, these results enable theselection of other antibodies based on the foregoing functionalproperties of E763 in addition to the structural properties that arealso discussed herein. The data also enables the potential study ofother therapeutic moieties in the treatment and eradication of tumors.

INCORPORATION BY REFERENCE

[0508] All references cited herein, including patents, patentapplications, papers, text books, and the like, and the references citedtherein, to the extent that they are not already, are herebyincorporated herein by reference in their entirety. In addition, thefollowing references are also incorporated by reference herein in theirentirety, including the references cited in such references:

[0509] Abertsen et al., “Construction and characterization of a yeastartificial chromosome library containing seven haploid human genomeequivalents.” Proc. Natl. Acad. Sci. 87:4256 (1990).

[0510] Anand et al., “Construction of yeast artificial chromosomelibraries with large inserts using fractionation by pulsed-field gelelectrophoresis.” Nucl. Acids Res. 17:3425-3433 (1989).

[0511] Berman et al. “Content and organization of the human Ig VH locus:definition of three new V_(H) families and linkage to the Ig C_(H)locus.” EMBO J. 7:727-738 (1988).

[0512] Brezinschek et al., “Analysis of the heavy chain repertoire ofhuman peripheral B-cells using single-cell polymerase chain reaction.”J. Immunol. 155:190-202 (1995).

[0513] Brownstein et al., “Isolation of single-copy human genes from alibrary of yeast artificial chromosome clones.” Science 244:1348-1351(1989).

[0514] Bruggeman et al. PNAS USA 86:6709-6713 (1989).

[0515] Bruggemann et al., “Human antibody production in transgenic mice:expression from 100 kb of the human IgH locus.” Eur. J Immunol.21:1323-1326 (1991).

[0516] Bruggeman, M. and Neuberger, M. S. in Methods: A companion toMethods in Enzymology 2:159-165 (Lemer et al. eds. Academic Press(1991)).

[0517] Bruggemann, M. and Neuberger, M. S. “Strategies for expressinghuman antibody repertoires in transgenic mice.” Immunology Today17:391-397 (1996).

[0518] Chen et al. “Immunoglobulin gene rearrangement in B-celldeficient mice generated by targeted deletion of the J_(H) locus”International Immunology 5:647-656 (1993)

[0519] Choi et al. “Transgenic mice containing a human heavy chainimmunoglobulin gene fragment cloned in a yeast artificial chromosome”Nature Genetics 4:117-123 (1993)

[0520] Coligan et al., Unit 2.1, “Enzyme-linked immunosorbent assays,”in Current protocols in immunology (1994).

[0521] Cook, G. P. and Tomlinson, I. M., “The human immunoglobulin V_(H)repertoire.” Immunology Today 16:237-242 (1995).

[0522] Cox et al., “A directory of human germ-line Vx segments reveals astrong bias in their usage.” Eur. J. Immunol. 24:827-836 (1994).

[0523] Dariavach et al., “The mouse IgH 3′-enhancer.” Eur. J. Immunol.21:1499-1504 (1991).

[0524] Den Dunnen et al., “Reconstruction of the 2.4 Mb human DMD-geneby homologous YAC recombination.” Human Molecular Genetics 1:19-28(1992).

[0525] Feeney, A. J. “Lack of N regions in fetal and neonatal mouseimmunoglobulin V-D-J junctional sequences.” J. Exp. Med. 172:137-1390(1990).

[0526] Fishwild et al., “High-avidity human IgGKκmonoclonal antibodiesfrom a novel strain of minilocus transgenic mice.” Nature Biotech.14:845-851 (1996).

[0527] Flanagan, J. G. and Rabbitts, T. H., “Arrangement of humanimmunoglobulin heavy chain constant region genes implies evolutionaryduplication of a segment containing g, e, and a genes.” Nature300:709-713 (1982).

[0528] Galfre, G. and Milstein, C., “Preparation of monoclonalantibodies: strategies and procedures.” Methods Enzymol. 73:3-46(1981).

[0529] Gemmill et al., “Protocols for pulsed field gel electrophoresis:Separation and detection of large DNA molecules.” Advances in GenomeBiology 1:217-251 (1991).

[0530] Gill et al., “Monoclonal anti-epidermal growth factor receptorantibodies which are inhibitors of epidermal growth factor binding andantagonists of epidermal growth factorstimulated tyrosine protein kinaseactivity.” J. Biol. Chem. 259:7755 (1984).

[0531] Green et al., “Antigen-specific human monoclonal antibodies frommice engineered with human Ig heavy and light chain YACs.” NatureGenetics 7:13-21 (1994).

[0532] Hermanson et al., “Rescue of end fragments of yeast artificialchromosomes by homologous recombination in yeast.” Nucleic Acids Res.19:4943-4948 (1991).

[0533] Huber et al., “The human immunoglobulin K locus. Characterizationof the partially duplicated L regions.” Eur. J. Immunol. 23:2860-2967(1993).

[0534] Jakobovits, A., “Humanizing the mouse genome.” Current Biology4:761-763 (1994).

[0535] Jakobovits, A., “Production of fully human antibodies bytransgenic mice.” Current Opinion in Biotechnology 6:561-566 (1995).

[0536] Jakobovits et al., “Germ-line transmission and expression of ahuman-derived yeast artificial-chromosome.” Nature 362:255-258 (1993).

[0537] Jakobovits, A. et al., “Analysis of homozygous mutant chimericmice: Deletion of the immunoglobulin heavy-chain joining region blocksB-cell development and antibody production.” Proc. Natl. Acad. Sci. USA90:2551-2555 (1993).

[0538] Kawamoto et al., “Growth stimulation of A431 cells by epidermalgrowth factor: Identification of high affinity receptors for EGF by ananti-receptor monoclonal antibody.” Proc. Nat. Acad. Sci., USA80:1337-1341 (1983).

[0539] Lonberg et al., “Antigen-specific human antibodies from micecomprising four distinct genetic modifications.” Nature 368:856-859(1994).

[0540] Lusti-Marasimhan et al., “Mutation of Leu25 and Val27 introducesCC chemokine activity into interleukin-8.” J. Biol. Chem. 270:2716-2721(1995).

[0541] Marks et al., “Oligonucleotide primers for polymerase chainreaction amplification of human immunoglobulin variable genes and designof family-specific oligonucleotide probes.” Eur. J. Immunol. 21:985-991(1991).

[0542] Matsuda et al., “Structure and physical map of 64 variablesegments in the 3′ 0.8-megabase region of the human immunoglobulinheavy-chain locus.” Nature Genetics 3:88-94 (1993).

[0543] Max, E. Molecular genetics of immunoglobulins. FundamentalImmunology. 315-382 (Paul, WE, ed., New York: Raven Press (1993)).

[0544] Mendez et al., “A set of YAC targeting vectors for theinterconversion of centric and acentric arms.” Cold Spring HarborLaboratory Press, Genome Mapping and Sequencing meeting, 163 (1993).

[0545] Mendez et al., “Analysis of the structural integrity of YACscomprising human immunoglobulin genes in yeast and in embryonic stemcells.” Genomics 26:294-307 (1995).

[0546] Ray, S. and Diamond, B., “Generation of a fusion partner tosample the repertoire of Splenic B-cells destined for apoptosis.” Proc.Natl. Acad. Sci. USA 91:5548-5551 (1994).

[0547] Sato et al., “Biological effects in vitro of monoclonalantibodies to human epidermal growth factor receptors” Mol. Biol. Med.1:511-529 (1983).

[0548] Schiestl, R. H. and Gietz, R. D., “High efficiency transformationof intact yeast cells using stranded nucleic acids as a carrier.” Curr.Genet. 16:339-346 (1989).

[0549] Sherman et al., “Laboratory Course Manual for Methods in YeastGenetics.” (Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.(1986)).

[0550] Silverman et al., “Meiotic recombination between yeast artificialchromosomes yields a single clone containing the entire BCL2protooncogene.” Proc. Natl. Acad. Sci. USA 87:9913-9917 (19______).

[0551] Srivastava, A. and Schlessinger, D., “Vectors for insertingselectable markers in vector arms and human DNA inserts of yeastartificial chromosomes (YACs).” Gene 103:53-59 (1991).

[0552] Taylor et al., “A transgenic mouse that expresses a diversity ofhuman sequence heavy and light chain immunoglobulins.” Nucleic AcidsResearch 20:6287-6295 (1992).

[0553] Taylor et al., “Human immunoglobulin transgenes undergorearrangement, somatic mutation and class switching in mice that lackendogenous IgM.” International Immunology 6:579-591 (1994).

[0554] Tuaillon et al., “Human immunoglobulin heavy-chain minilocusrecombination in transgenic mice: gene-segment use in m and gtranscripts.” Proc. Natl. Acad. Sci. USA 90:3720-3724 (1993).

[0555] Tuaillon et al. “Analysis of direct and inverted DJHrearrangements in a human Ig heavy chain transgenic minilocus” J.Immunol. 154:6453-6465 (1995)

[0556] Vaughan et al., “Human antibodies with subnanomolar affinitiesisolated from a large non-immunized phage display library.” NatureBiotech. 14:309-314 (1996).

[0557] Wagner et al., “The diversity of antigen-specific monoclonalantibodies from transgenic mice bearing human immunoglobulin geneminiloci.” Eur. J. Immunol. 24:2672-2681 (1994).

[0558] Weichhold et al., “The human immunoglobulin κ locus consists oftwo copies that are organized in opposite polarity.” Genomics 16:503-511(1993).

[0559] Yamada, M. et al., “Preferential utilization of specificimmunoglobulin heavy chain diversity and joining segments in adult humanperipheral blood B lymphocytes.” J. Exp. Med. 173:395-407 (1991).

[0560] 1. Ullrich, A. and Schlessinger, J. Signal transduction byreceptors with tyrosine kinase activity. (Review). Cell, 61: 203-212,1990.

[0561] 2. Baselga, J. and Mendelsohn, J. Receptor blockade withmonoclonal antibodies as anti-cancer therapy. (Review). Pharmacol Ther,64: 127-154, 1994.

[0562] 3. Mendelsohn, J. and Baselga, J. Antibodies to growth factorsand receptors. (Review). In: V. T. DeVita, S. Hellman and S. A.Rosenberg (eds.), Biologic Therapy of Cancer, pp. 607-623, Philadelphia:J. B. Lippincott Company. 1995.

[0563] 4. Fan, Z. and Mendelsohn, J. Therapeutic application ofanti-growth factor receptor antibodies. (Review). Curr Opin Oncol, 10:67-73, 1998.

[0564] 5. Riedel, H., Massoglia, S., Schlessinger, J., and Ullrich, A.Ligand activation of overexpressed epidermal growth factor receptorstransforms NIH 3T3 mouse fibroblasts. Proc Natl Acad Sci USA, 85:1477-1481, 1988.

[0565] 6. Modjtahedi, H. and Dean, C. The receptor for EGF and itsligands: expression, prognostic value and target for therapy in cancer(Review). Intl J of Oncology, 4: 277-296, 1994.

[0566] 7. Gullick, W. J. Prevalence of aberrant expression of theepidermal growth factor receptor in human cancers. Br Medical Bulletin,47: 87-98, 1991.

[0567] 8. Salomon, D.S., Brandt, R., Ciardiello, F., and Normanno, N.Epidermal growth factor-related peptides and their receptors in humanmalignancies. Crit Rev Oncol Hematol, 19: 183-232, 1995.

[0568] 9. Aboud-Pirak, E., Hurwitz, E., Pirak, M. E., Bellot, F.,Schlessinger, J., and Sela, M. Efficacy of antibodies to epidermalgrowth factor receptor against KB carcinoma in vitro and in nude mice. Jof the National Cancer Inst, 80: 1605-1611, 1988.

[0569] 10. Modjtahedi, H., Styles, J. M., and Dean, C. J. The human EGFreceptor as a target for cancer therapy: six new rat mAbs against thereceptor on the breast carcinoma MDA-MB 468. Br J Cancer, 67: 247-253,1993.

[0570] 11. Modjtahedi, H., Eccles, S., Box, G., Styles, J., and Dean, C.Immunotherapy of human tumour xenografts overexpressing the EGF receptorwith rat antibodies that block growth factor-receptor interaction. Br JCancer, 67: 2611993.

[0571] 12. Fan, Z., Baselga, J., Masui, H., and Mendelsohn, J. Antitumoreffect of anti-epidermal growth factor receptor monoclonal antibodiesplus cis-diamminedichloroplatinum on well established A431 cellxenografts. Cancer Research, 53: 4637-4642, 1993.

[0572] 13. Baselga, J., Norton, L., Masui, H., Pandiella, A., Coplan,K., Miller, W. H., and Mendelsohn, J. Antitumor effects of doxorubicinin combination with anti-epidermal growth factor receptor monoclonalantibodies. J of the National Cancer Inst, 85: 1327-1333, 1993.

[0573] 14. Goldstein, N.I., Prewett, M., Zuklys, K., Rockwell, P., andMendelsohn, J. Biological efficacy of a chimeric antibody ot theepidermal growth factor receptor in a human tumor xenograft model.Clinical Cancer Research, 1: 1311-1318, 1995.

[0574] 15. Prewett, M., Rockwell, R., Rockell, R. F., Giorgio, N. A.,Mendelsohn, J., Scher, H. I., and Goldstein, N. I. The biologic effectsof C225, a chimeric monoclonal antibody to the EGFR, on human prostatecarcinoma. J Immunother Emphasis Tumor Immunol, 19: 419-427, 1996.

[0575] 16. Slovin, S. F., Kelley, W. K., Cohen, R., Cooper, M., Malone,T., Weingard, K., Waksal, H., Falcey, J., Mendelsohn, J., and Scher, H.I. Epidermal gowth factor receptor (EGF-r) monoclonal antibody (MoAb)C225 and doxorubicin (DOC) in androgen-independent (AI) prostate cancer(PC): results of a phase Ib/IIa study. Proc Am Soc Clin Oncol, 16:311al997.(Abstract)

[0576] 17. Falcey, J., Pfister, D., Cohen, R., Cooper, M., Bowden, C.,Burtness, B., Mendelsohn, J., and Waksal, H. A study of anti-epidermalgrowth factor receptor (EGFr) monoclonal antibody C225 and cisplatin inpatients (pts) with head and neck or lung carcinomas. Proc Am Soc ClinOncol, 16: 383a1997.(Abstract)

[0577] 18. Mendez, M. J., Green, L. L., Corvalan, J. R. F., Jia, X.-C.,Maynard-Currie, C. E., Yang, X. -D., Gallo, M. L., Louie, D. M., Lee, D.V., Erickson, K. L., Luna, J., Roy, C. M. -N., Abderrahim, H.,Kirschenbaum, F., Noguchi, M., Smith, D. H., Fukushima, A., Hales, J.F., Finer, M. H., Davis, C. G., Zsebo, K. M., and Jakobovits, A.Functional transplant of megabase human immunoglobulin locirecapitulates human antibody response in mice. Nat Gen, 15: 146-156,1997.

[0578] 19. Jakobovits, A. The long-awaited magic bullets: therapeutichuman monoclonal antibodies from transgenic mice. Exp Opin Invest Drugs,7: 607-614, 1998.

[0579] 20. Debanne, M. T., Pacheco-Oliver, M. C., and O'Connor-McCourt,M. D. Purification of the extracellular domain of the epidermal growthfactor receptor produced by recombinant baculovirus-infected insectcells in a 10-L reactor. In: C. D. Richardson (ed.), BaculovirusExpression Protocols, pp. 349-361, Totowa, N.J.: Humana Press Inc. 1995.

[0580] 21. Ishiyama, M., Tominaga, H., Shiga, M., Sasamoto, K., Ohkura,Y., and Ueno, K. A combined assay of cell viability and in vitrocytotoxicity with a highly water-soluable tetrazolium salt, neutral redand crystal violet. Biol Pharm Bull, 19: 1518-1520, 1996.

[0581] 22. McConnell, H. M., Owicki, J. C., Parce, J. W., Miller, D. L.,Baxter, G. T., Wada, H. G., and Pitchford, S. The cytosensormicrophysiometer: biological applications of silicon technology.Science, 257: 1906-1912, 1992.

[0582] 23. Bogovski, P. Tumours of the skin. In: V.S. Turusov and U.Mohr (eds.), Tumours of Mouse, pp. 1-26, Lyon: IARC ScientificPublications. 1994.

[0583] 24. Ennis, B. W., Valverius, E. M., Bates, S. E., Lippman, M. E.,Bellot, F., Kris, R., Schlessinger, J., Masui, H., Goldenberg, A.,Mendelsohn, J., and Dickson, R. B. Anti-epidermal growth factor receptorantibodies inhibit the autocrine-stimulated growth of MDA-468 humanbreast cancer cells. Molecular Endocrinology, 3: 1830-1838, 1989.

[0584] 25. Kawamoto, T., Mendelsohn, J., Gordon, A. L., Sato, G. H.,Lazar, C. S., and Gill, G. N. Relation of epidermal growth factorreceptor concentration to growth of human epidermoid carcinoma A431cells. J of Biological Chemistry, 259: 7761-7766, 1984.

[0585] 26. Masui, H., Moroyama, T., and Mendelsohn, J. Mechanism ofantitumor activity in mice for anti-epidermal growth factor receptormonoclonal antibodies with different isotypes. Cancer Research, 46:5592-5598, 1986.

[0586] 27. Wu, X., Fan, Z., Masui, H., Rosen, N., and Mendelsohn, J.Apoptosis induced by an anti-epidermal growth factor receptor monoclonalantibody in human colorectal carcinoma cell line and its delay byinsulin. J Clin Invest, 95: 1897-1905, 1995.

[0587] 28. Sturgis, E. M., Sacks, P. G., Masui, H., Mendelsohn, J., andSchantz, S. P. Effects of antiepidermal growth factor receptor antibody528 on the proliferation and differentiation of head and neck cancer.Otolaryngol Head Neck Surg, 111: 633-643, 1994.

[0588] 29. Modjtahedi, H., Eccles, S., Sandle, J., Box, G., Titley, J.,and Dean, C. Differentiation or immune destruction: two pathways fortherapy of squamous cell carcinomas with antibodies to the epidermalgrowth factor receptor. Cancer Res, 54: 1695-1701, 1994.

[0589] 30. Viloria Petit, A. M., Rak, J., Hung, M. -C., Rockwell, P.,Goldstein, N., Fendly, B., and Kerbel, R. S. Neutralizing antibodiesagainst epidermal growth factor and ErbB-2/neu receptor tyrosine kinasesdown-regulate vascular endothelial growth factor production by tumorcells in vitro and in vivo: angiogenic implications for signaltransduction therapy of solid tumors. Am J Pathol, 151: 1523-1530, 1997.

[0590] 31. Kita, Y., Tseng, J., Horan, T., Wen, J., Philo, J., Chang,D., Ratzkin, B., Pacifici, R., Brankow, D., Hu, S., Luo, Y., Wen, D.,Arakawa, T., and Nicolson, M. ErbB receptor activation, cell morphologychanges, and apoptosis induced by anti-Her2 monoclonal antibodies.Biochem Biophys Res Commun, 226: 59-69, 1996.

[0591] 32. Slamon, D., Leyland-Jones, B., Shak, S., Paton, V.,Bajamonde, A., Fleming, T., Eiermann, W., Wolter, J., Baselga, J., andNorton, L. Addition of Herceptin™ (human anti-Her2 antibody) to firstline chemotherapy for HER2 overexpressing metastatic breast cancer(Her2+/mbc) markedly increases anticancer activity: a randomized,multinational controlled phase III trial. Proc of ASCO, 17: 98a,1998.(Abstract)

[0592] 33. Cobleigh, M. A., Bogel, C. L., Tripathy, D., Robert, N. J.,Scholl, S., Fegrenbacher, L., Paton, V., Shak, S., Lieberman, G., andSlamon, D. Efficacy and safety of Herceptin™ (humanized anti-Her2antibody) as a single agent in 222 women with Her2 overexpression whorelapsed following chemotherapy for metastatic breast cancer. Proc ofASCO, 17: 97a, 1998.(Abstract)

Equivalents

[0593] The foregoing description and Examples detail certain preferredembodiments of the invention and describes the best mode contemplated bythe inventors. It will be appreciated, however, that no matter howdetailed the foregoing may appear in text, the invention may bepracticed in many ways and the invention should be construed inaccordance with the appended claims and any equivalents thereof.

What we claim is:
 1. An antibody that binds to epidermal growth factorreceptor, that is characterized by the following functions: Inhibitstyrosine phosphorylation of EGF-r; Is internalized with EGF-r; Inhibitsthe degradation of EGF-r; and Inhibits the EGF induced degradation ofEGF-r.
 2. An antibody that binds to epidermal growth factor receptor,that is characterized by the following functions: Protects threoninephosporylation of EGF-r.
 3. An antibody that binds to epidermal growthfactor receptor, that is characterized by the following functions:Protects threonine phosphorylation of a 63 KD protein.
 4. An antibodythat binds to epidermal growth factor receptor, that is characterized bythe following functions: Inhibits VEGF production by tumor cells bygreater than 50%; and Inhibits VEGF production by endothelial cells bygreater than 40%.
 5. The antibody of claim 4, wherein the tumor cellsare A431 cells.
 6. The antibody of claim 4, wherein the tumor cells areECV304 cells.
 7. An antibody that binds to epidermal growth factorreceptor, that is characterized by the following functions: Inhibitstyro sine phosphorylation of EGF-r; Is internalized with EGF-r; Inhibitsthe degradation of EGF-r; Inhibits the EGF induced degradation of EGF-r;Protects threonine phosporylation of EGF-r; Protects threoninephosphorylation of a 63 KD protein; Inhibits VEGF production by tumorcells by greater than 50%; and Inhibits VEGF production by endothelialcells by greater than 40%.